TGGE Manual

TGGE Manual

Biometra
a Whatman company
TGGE System
230 V
115 V
Code-No. 024-000
Code-No. 024-090
Manual
March 1999
!! Warning !!
Please read these instructions carefully
before using this apparatus!
Biometra biomedizinische Analytik GmbH
Rudolf-Wissell-Straße 30, D-37079 Göttingen
P.O. Box 1544, D-37005 Göttingen
Tel:++49 – (0)5 51 / 50 68 6-0 * Fax: ++49 – (0)5 51 / 50 68 6-66
e-mail: [email protected]
internet: http://www.biometra.de
TGGE System
The TGGE method is covered by patents issued to Diagen (now QIAGEN GmbH).
The polymerase chain reaction (PCR) process is covered by patents issued to
Hoffmann-La Roche.
Acryl-Glide is a trademark of Amresco Inc.
Biometra is a trademark of Biometra GmbH.
Whatman is a trademark of Whatman International Ltd.
The POLAND software service established by Gerhard Steger, Department of
Biophysics, University of Duesseldorf, is available by internet http://www.biophys.uniduesseldorf.de/service/polandform.html.
Manual Version 2.5
Software Version 2.10, 2.11

Biometra GmbH, Rudolf-Wissell-Str. 30, 37079 Göttingen, Germany
7/24/2002
TGGE System
1
Table of Contents
1
The TGGE System .............................................................................................. 4
1.1
Introduction .................................................................................................... 4
1.2
Principle of method ........................................................................................ 4
1.3
Special features of the TGGE System ........................................................... 5
1.4
How to start with TGGE analysis ................................................................... 6
2
General Recommendations ............................................................................... 8
2.1
Safety warnings ............................................................................................. 8
2.2
Notes for use.................................................................................................. 8
3
Components of the TGGE System .................................................................... 9
3.1
Electrophoresis unit ..................................................................................... 10
3.2
Controller unit............................................................................................... 17
3.2.1
Instrument keys and ports ...................................................................... 17
3.2.2
Programming of the TGGE Controller..................................................... 18
4
Sample preparation .......................................................................................... 24
4.1
Purity of samples ......................................................................................... 24
4.2
Quantity and volumes of samples ................................................................ 25
5
Setting up polyacrylamide gels....................................................................... 25
5.1
Selecting Concentration of PAA gels ........................................................... 25
5.2
Setting up the gel solution............................................................................ 25
5.3
Some remarks corresponding to standard TGGE conditions ....................... 28
5.4
Assembling the gel sandwich....................................................................... 30
5.5
Disassembling the gel sandwich .................................................................. 32
6
Electrophoresis with the TGGE System ......................................................... 33
6.1
Electrophoresis conditions ........................................................................... 33
6.2
Preparing the electrophoresis unit ............................................................... 33
6.3
Perpendicular TGGE.................................................................................... 35
6.4
Parallel TGGE.............................................................................................. 39
6.5
Silver staining............................................................................................... 44
6.6
Ethidium bromide-staining ........................................................................... 48
6.7
Blotting......................................................................................................... 48
6.8
Autoradiography........................................................................................... 48
6.9
Elution of DNA from the TGGE gel .............................................................. 48
7
TGGE in analysis of point mutations in dsDNA ............................................ 49
7.1
....... Theoretical background of a detection rate approximating 100% for point
mutations - calculations with the POLAND program .................................... 49
7.2
The “old” Poland program (Old Server)........................................................ 51
7.2.1
About the Poland service........................................................................ 51
7.2.2
Program-specific information .................................................................. 51
7.2.3
How to use the “old” Poland program (Standard) ................................... 53
7.3
The “new” Poland program (New Server) .................................................... 55
7.3.1
About the Poland service........................................................................ 55
7.3.2
Program-specific information .................................................................. 55
7.3.3
References for Poland Service ............................................................... 56
7.3.4
HELP for Poland Service: ....................................................................... 56
TGGE System
2
7.3.5
How to use the “new” Poland program ................................................... 65
7.4
The optimized DNA fragment....................................................................... 66
7.4.1
Asymmetric GC-clamps for PCR primers used for TGGE analysis......... 68
7.4.2
Chemical clamp with Psoralen (Furo[3,2-g]coumarin, C11H6O3)............. 68
7.4.3
POLAND analysis of samples................................................................. 69
8
Optimizing parallel TGGE by perpendicular TGGE....................................... 70
8.1
Check short DNA fragments for their melting behavior ................................ 70
8.2
From perpendicular to parallel TGGE .......................................................... 71
9
TGGE / SSCP..................................................................................................... 72
9.1
Running an SSCP on the TGGE.................................................................. 72
9.2
DNA sample preparation.............................................................................. 73
9.3
Gel casting ................................................................................................... 73
9.4
Electrophoresis ............................................................................................ 75
9.5
Routine analysis........................................................................................... 75
10 TGGE in RNA analysis ..................................................................................... 76
10.1 Completely double-stranded RNA................................................................ 76
10.2 Partly double-stranded RNA, e.g. viroid RNA .............................................. 76
10.3 ...........Single-stranded RNA with single hairpin structures, m-RNA secondary
structures ..................................................................................................... 76
10.4 Staining........................................................................................................ 77
11 TGGE in protein analysis ................................................................................. 78
11.1 Buffers ......................................................................................................... 78
12 Trouble-shooting .............................................................................................. 80
13 TGGE Testkit..................................................................................................... 93
13.1 Introduction .................................................................................................. 93
13.2 Protocol........................................................................................................ 93
13.2.1 Gel composition:..................................................................................... 93
13.2.2 Running buffer: ....................................................................................... 94
13.2.3 Electrophoresis parameters:................................................................... 95
13.3 Gel images................................................................................................... 95
14 Appendix ........................................................................................................... 96
14.1 Technical Data ............................................................................................. 96
14.1.1 System.................................................................................................... 96
14.1.2 Electrophoresis Chamber ....................................................................... 96
14.1.3 TGGE System Controller with integrated power pack............................. 96
14.2 Buffers ......................................................................................................... 97
14.3 Silver staining solutions: .............................................................................. 99
15 References ...................................................................................................... 101
16 Order information and spare parts................................................................ 105
17 Instructions for return shipment ................................................................... 106
TGGE System
3
18 Equipment Decontamination Certificate....................................................... 107
19 Warranty .......................................................................................................... 108
Declaration of Conformity
4
TGGE System
1
The TGGE System
1.1 Introduction
Temperature Gradient Gel Electrophoresis is a new and powerful electrophoresis
method for separation of nucleic acids like DNA or RNA or for analysis of proteins.
The TGGE method, which is covered by patents, uses the temperature dependent
change of conformation for separating molecules (for review see Reference 1).
Since the introduction of the first commercial available TGGE apparatus in 1989
temperature gradient gel electrophoresis has gained high interest in scientific and
clinical laboratories due to the unprecedented resolution capability and easiness of
analysis. The range of scientific publications using the TGGE method is broad and
covers all disciplines which use molecular biology methods: e.g. Oncology2-4,
Virology5,6, Immunology7,8, RNA Viroid Research9-12, Prion Research13, Population
Analysis14-15. The TGGE method has also been used for quantitative analysis in
industry16-17 and for conformational analysis of proteins18-19.
1.2 Principle of method
Conventional protein or nucleic acid electrophoresis separates molecules mainly
according to size or charge. TGGE adds a new parameter for separation, namely the
shape of the molecule.
The shape is mostly determined by the secondary and tertiary structure of the
molecule and can be changed by external influences like temperature, salt
concentration, pH etc.
The conformation both of proteins and nucleic acids depend on weak binding forces
like hydrogen bonds or van der Waals bonds. Increasing the temperature above a
certain limit breaks down these bonds. The molecules will adopt a so called
denatured conformation in contrast to the native one.
E.g. with DNA it is possible to determine the temperature which is necessary to break
down hydrogen bonds along double stranded DNA. This temperature is called
midpoint of transition (TM) or melting temperature and characteristic for a certain
stretch of DNA (see figure 1). TGGE uses the melting temperature to identify DNA
which differs in sequence among a mixture of molecules of the same size.
TGGE therefore not only separates molecules but gives additional information about
the sequence (DNA or RNA) or the stability of proteins.
Migration
branched DNA (partially denatured conf.)
single stranded DNA
(completely denatured)
double
stranded DNA
T1 (cold)
TM
Temperature
T2 (warm)
Figure 1: Schematic drawing of different conformations of DNA during temperature gradient
gel electrophoresis.
5
TGGE System
1.3 Special features of the TGGE System
The microprocessor controlled gradient block of the TGGE System allows strictly
defined linear gradients with high resolution. A run distance of 2 mm which can easily
be detected by eye corresponds to a maximum temperature difference of about
0.6°C. Therefore even slightest differences of molecules can be detected by the new
TGGE System.
Because of the small amount of material used for separation DNA or RNA fragments
appear as fine bands which can clearly be distinguished from each other. Even
complex band patterns can be analyzed due to the high resolution capability of the
gradient block.
Comparing the TGGE method with another screening method like SSCP shows
superior performance of the TGGE method20-22.
The controlled temperature conditions make repetition of experiments easy and lead
to reproducible gel results. The small format of the gradient block has been optimized
in order to reduce sample volume and especially to save experimental time.
Perpendicular and parallel TGGE are two different modes applicable with the
Biometra TGGE System without need for specialized parts or equipment.
Whereas perpendicular TGGE is mostly used for defining optimal separation
conditions, parallel TGGE allows the analysis of multiple samples (e.g. screening).
perpendicular TGGE:
temperature
gradient
is
perpendicular
to
the
electrophoretic run direction
→ one sample is spread over a broad temperature range
parallel TGGE:
temperature gradient is parallel to run direction
→ multiple samples are spread over a narrow temp. range
Perpendicular TGGE:
T1 (cold)
-
Parallel TGGE
T2 (warm)
-
T1 (cold)
Gel slots
Run
direction
+
+
T2 (warm)
Figure 2: Schematic drawing of typical results after perpendicular TGGE (left panel)
and parallel TGGE (right panel).
6
TGGE System
1.4 How to start with TGGE analysis
Starting with the gene of interest one develops the right combination of PCR primers
for amplification of the desired gene fragment. The polymorphic site must be located
inside the amplicon and not at the far end. The POLAND software helps one to
identify suitable primers and the adequate fragment length of the amplicon. This
program gives a rough estimation too, what temperature parameters will fit best to
the desired separation23-25.
A revised version of the POLAND program can be found on the world wide web
(http:// www.biophys.uni-duesseldorf.de/service/polandform.html).
For example the polymorphic site is represented by allele A and allele a. The two
alleles can either exist as homoduplices (AA or aa) or heteroduplices (Aa or aA) (see
figure 3).
A A
A a
a a
Sequence of
Allele A
Sequence of
Allele a
A
A
a
a
A
C
C
G
T
G
G
C
A
T
C
G
T
A
G
C
a A
Figure 3: Schematic drawing of double stranded DNA with polymorphism „A“ or „a“
(left panel) and corresponding DNA sequence (right panel). Each line represents
double stranded DNA.
The TGGE System used as perpendicular TGGE (see above, figure 2) gives the
possibility to identify the different alleles by their individual melting behavior. Samples
with homoduplex „AA“ or „aa“ have a distinct melting temperature (e-g. Tm1 and Tm2,
see figure 4), at which double stranded DNA separates into branched DNA. At even
higher temperature the branched DNA separates into individual strands.
After perpendicular TGGE, a heteroallele sample like „Aa“ normally shows four
different Tm values. The PCR amplification of a heteroallele sample results in four
different double stranded DNA types: The two homoduplices "AA" and "aa" as well as
two heteroduplices "Aa" and "aA", which have a non-pairing base at the polymorphic
site. This non-pairing base will lead to a shift of the Tm to lower values (Tm3 and Tm4).
Perpendicular TGGE shows at which temperature the different DNA strands will
separate. For future analysis by perpendicular or parallel TGGE a narrower
temperature range which includes the TM values of homo- and heteroduplices can be
used.
Please remember always, that wild type and mutant DNA have to be mixed before
PCR to get the 4 bands with different melting behavior. Sometimes the melting
difference between heteroduplices cannot be resolved but remember that 3 bands on
the TGGE gel are enough to detect a mutation.
7
TGGE System
T1 (cold)
T2 (warm)
_
Gel slot
+
Tm4: Heteroduplex aA
Tm1: Homoduplex AA
Tm3: Heteroduplex Aa
Tm2: Homoduplex aa
Figure 4: Schematic drawing of melting behavior of double stranded, heterozygotic
DNA with allele typ „Aa“ after perpendicular TGGE.
Screening of multiple samples is performed by using parallel TGGE (see above,
figure 2). Parallel TGGE looks like conventional SSCP-analysis, but has a higher
probability to identify possible mutants.
TM4: Heteroduplex aA
-
T1 (cold)
TM3: Heteroduplex Aa
TM2: Homoduplex aa
Tm1: Homoduplex AA
+
T2 (warm)
Figure 5: Schematic drawing of a screening for double stranded, heterozygotic DNA
with allele type „Aa“ after parallel TGGE. (Sometimes the melting difference between
heteroduplices can not be resolved.)
Different plates with pre-fixed slots for 8, 12 or 18 samples are available for
screening purposes (see chapters 6.4 and 15).
TGGE System
8
2 General Recommendations
2.1 Safety warnings
Check the voltage of the power supply and of the control unit before
use.
In any case of malfunction of the power supply, controller or
electrophoresis chamber, do not open the case but contact Biometra or
your local distributor.
First switch off the power switch of the power supply before opening the
lid of the electrophoresis chamber if you want to interrupt or stop the
electrophoresis run.
During electrophoresis don’t touch the electrode wires or the buffer
inside the electrophoresis chamber. High voltage. Danger of life!
Whenever polyacrylamide gels are handled pay attention to standard
laboratory safety regulations, e.g. wear lab coat, protective gloves and
eye shield. Polyacrylamide is neurotoxic.
2.2 Notes for use
•
•
•
•
Do not scratch the protective foil of the gradient block. In case of a damaged foil
contact Biometra or your local distributor for a replacement foil.
Do not use strong acids or basic solutions or organic solvents for cleaning glass
plates, the electrophoresis chamber or the gradient block.
Do not incubate glass plates over night in cleaning solution.
Wear protective gloves during all steps of the silver staining protocols to avoid
staining artifacts due to the high sensitivity of the staining protocol.
9
TGGE System
3 Components of the TGGE System
The TGGE System contains all components which are necessary to get started. All
kinds of TGGE applications (parallel or perpendicular TGGE, Constant Temperature
GE, Time resolved TGGE) can be run with the System. For certain applications
which need different numbers or sizes of sample slots the adequate parts are
available and can be ordered from Biometra or your local distributor (see 6.3. Order
Information).
The TGGE System (Order number: 024-000) consists of:
• TGGE Controller with integrated power supply, 100 program
stores and control function of temperature and electrophoresis
conditions
• TGGE-Electrophoresis unit with 2 removable buffer
chambers, Peltier-element powered gradient block and control
cable
• TGGE Starter Kit contains
3 plane „Bonding“ glass plates
1 glass plate with 8 slots for parallel TGGE
1 glass plate with 1 rectangular slot for
perpendicular TGGE
1 glass pate with 12 slots for parallel TGGE
Electrophoresis wicks (100/pkg)
Polybond film (25/pkg)
1 Cover glass plate and 10 cover films
1 Acryl-Glide (100 ml)
• Manual
024-001
024-002
024-003
024-021
024-022
024-023
024-025
024-015
024-030
024-031
211-319
When unpacking your System please check whether all mentioned parts are
included. If individual parts are missing call Biometra or your local distributor.
10
TGGE System
3.1 Electrophoresis unit
The electrophoresis unit consists of 4 parts:
n 1 safety lid with 2 electric plugs (anode and cathode)
o 2 removable electrophoresis chambers each with platinum wires and electric connectors (volume: max. 250 ml)
➌ housing with Peltier-element powered gradient block
q 37 pin connecting cable to control unit
n
o
➌
Figure 6: Parts of the electrophoresis unit
11
TGGE System
T1
L1
L2
L3
L4
L5
L6
T2
T
1
L
1
L
2
L
3
L
4
L
5
L
6
T1
L1
L2
L3
L4
L5
L6
T2
T
2
Figure 6b: Gradient block with temperature lines and marks for positioning the gel
slots (top). Safety lid with arrow indicating the running direction of nucleic acids
(middle). Gradient block covered with safety lid; setup for a perpendicular TGGE run
(bottom, left); setup for a parallel TGGE run (bottom, right).
12
TGGE System
The gradient block is centrally positioned in the middle of the electrophoresis unit and
protected by a layer of white foil. This foil is necessary to protect the electronic parts
beneath it from liquid, buffer or other harmful chemicals.
If the protection foil has been scratched during use
stop working and
exchange the protection foil with a new one.
The two opposite sides of the gradient block are marked with lettering T1 and T2.
Beneath these symbols the Peltier-elements which build up the temperature gradient
during electrophoresis can be found. Both sides of the gradient block can reach any
preset temperature from 15°C - 80°C. The orientation of the temperature gradient,
i.e. which side of the gradient shall be cold or hot, can be freely determined.
Between symbols T1 and T2 six thick lines (L1 to L6) and five thin lines (not coded)
are marked on the block, which represent the entire linear range of the gradient block
(see figure 7). The temperature difference between two lines is identical from line to
line. E.g. if T1 is 30°C and T2 is 75°C, the temperature difference is 7.4°C between
two thick lines or 3.7°C between a thick and a thin line.
T1
L1
L2
L3
L4
L5
L6
T2
When performing parallel TGGE the beginning and the end of the linear temperature
gradient are represented by the first and the last line. Whereas when performing
perpendicular TGGE the ends of each line represent the linear temperature range.
When choosing a temperature gradient e.g. between 25°C and 65°C these two
temperatures can actually be found at the first marked line (figure 7: 10mm distance
to the block edge) and at the last marked line (figure 7: 50 mm distance) on the
block. The block areas to the left and right of these lines are slightly hotter
respectively cooler (see figure 7 and table 1 + 2).
13
TGGE System
Linearity of block
80
Temperature (°C)
70
60
50
40
30
20
10
0
1
6
11
16
21
26
31
36
41
46
51
56
61
Distance (mm)
T1
L1
L2
L3
L4
L5
L6
T2
Figure 7: upper panel: Temperature profile of gradient block measured by a micro
sensor on top of the gradient plate every 1mm beginning from one edge of the block.
10 mm distance and 50 mm distance correspond to first (L1) and last marked thick
line (L2) on the gradient block respectively.
lower panel: Schematic drawing of the block.
14
TGGE System
The maximum temperature difference between the two sides of the gradient block (T1
and T2) during electrophoresis is 45 Kelvin. That means it is possible to build up a
gradient between 35°C and 80°C or between 25°C and 70°C, just to give two
examples.
----------------------------------------------------------------------------------------------------------------Programming T1 and T2, the actual temperature of L1 to L6 can be calculated by the following
formula:
Ln = L1 + (n - 1) ∆ϑ
(Ln = Temperature of line n; n = 1 .......6)
L6 - L1
∆ϑ = ------------
(∆ϑ = Temperature difference between two thick lines)
5
T1
L1
L2
L3
L4
L5
L6
T2
----------------------------------------------------------------------------------------------------------------When leaving out the gradient function the block can be cooled down to 4°C or
heated up to 80°C. Although Peltier elements reach lower respectively higher
temperature values, the surrounding plastic material does not permit the temperature
range of the TGGE System to be extended.
The electrophoresis buffer chambers can freely be positioned around the gradient
block. This makes it easy to switch between parallel or perpendicular TGGE. When
the electrophoresis buffer chambers stay parallel to the lines of the gradient block
(see figure 8 left panel) parallel TGGE applications can be run. By simply switching
the chambers perpendicular to the lines of the gradient block (see figure 8 right
panel) it is possible to run perpendicular TGGE.
T1
L1
L2
L3
L4
L5
L6
T2
T1
L1
L2
L3
L4
L5
L6
T2
Figure 8: Orientation of the two electrophoresis buffer chambers (- represents
cathode, + represents anode) relative to the centrally positioned gradient block.
15
TGGE System
T1
15,94
16,94
17,94
18,94
19,94
20,94
21,94
22,94
23,94
24,94
25,94
26,94
27,94
28,94
29,94
30,94
31,94
32,94
33,94
34,94
35,94
36,94
37,94
38,94
Programming L1 and L6 (°C):
L1
L2
L3
L4
L5
L6
T2
DELTA
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
61,06
62,06
63,06
64,06
65,06
66,06
67,06
68,06
69,06
70,06
71,06
72,06
73,06
74,06
75,06
76,06
77,06
78,06
79,06
80,06
81,06
82,06
83,06
84,06
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
7,4
27,4
28,4
29,4
30,4
31,4
32,4
33,4
34,4
35,4
36,4
37,4
38,4
39,4
40,4
41,4
42,4
43,4
44,4
45,4
46,4
47,4
48,4
49,4
50,4
34,8
35,8
36,8
37,8
38,8
39,8
40,8
41,8
42,8
43,8
44,8
45,8
46,8
47,8
48,8
49,8
50,8
51,8
52,8
53,8
54,8
55,8
56,8
57,8
42,2
43,2
44,2
45,2
46,2
47,2
48,2
49,2
50,2
51,2
52,2
53,2
54,2
55,2
56,2
57,2
58,2
59,2
60,2
61,2
62,2
63,2
64,2
65,2
49,6
50,6
51,6
52,6
53,6
54,6
55,6
56,6
57,6
58,6
59,6
60,6
61,6
62,6
63,6
64,6
65,6
66,6
67,6
68,6
69,6
70,6
71,6
72,6
Table 1: Examples for actual temperatures on the gradient block programming L1
and L6
16
TGGE System
Programming T1 and T2 (°C):
T1
L1
L2
L3
L4
L5
L6
T2
DELTA
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
24,05
25,05
26,05
27,05
28,05
29,05
30,05
31,05
32,05
33,05
34,05
35,05
36,05
37,05
38,05
39,05
39,96
40,87
41,78
42,69
43,6
31,43
32,43
33,43
34,43
35,43
36,43
37,43
38,43
39,43
40,43
41,43
42,43
43,43
44,43
45,43
46,43
47,18
47,92
48,67
49,41
50,16
38,81
39,81
40,81
41,81
42,81
43,81
44,81
45,81
46,81
47,81
48,81
49,81
50,81
51,81
52,81
53,81
54,39
54,97
55,56
56,14
56,72
46,19
47,19
48,19
49,19
50,19
51,19
52,19
53,19
54,19
55,19
56,19
57,19
58,19
59,19
60,19
61,19
61,61
62,03
62,44
62,86
63,28
53,57
54,57
55,57
56,57
57,57
58,57
59,57
60,57
61,57
62,57
63,57
64,57
65,57
66,57
67,57
68,57
68,82
69,08
69,33
69,59
69,84
60,95
61,95
62,95
63,95
64,95
65,95
66,95
67,95
68,95
69,95
70,95
71,95
72,95
73,95
74,95
75,95
76,04
76,13
76,22
76,31
76,4
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
80
80
80
80
80
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,38
7,216
7,052
6,888
6,724
6,56
Table 1: Examples for actual temperatures on the gradient block programming T1
and T2
17
TGGE System
3.2 Controller unit
The controller is a highly integrated, micro processor driven unit for controlling the
temperature, ramping time and ramping rate of the gradient block as well as
supplying the power for the electrophoresis unit. For entering and storing run
parameters the front panel of the controller offers 4 function keys and a full numerical
key pad. During the run the display of the controller continuously shows the current
parameters.
3.2.1 Instrument keys and ports
p
o
n
n Power on/off switch
o Display
p 4 function keys A, B, C, D
q Alphanumerical key pad
q
Figure 9: Front panel of the TGGE System Controller.
n
o
p
q
r
n Computer port (RS 232)
o Connectors for electrode cable:
red = anode, black = cathode
p Printer port
q Interface to electrophoresis unit
r Mains and fuses
Figure 10: TGGE System Controller from the rear
TGGE System
18
3.2.2 Programming of the TGGE Controller
After switching on the controller the display shows the instruments name and the
software version. Immediately afterwards the main menu appears.
Main menu
T1: 22.0°C
T2: 22.0°C
block off
A? B Elpho
+
C programs D
At the bottom line of the display 4 possible options which can be
retrieved
by the 4 functions keys ªA, B, C, D are shown. These 4 options
change
during programming relative to the chosen menu.
(Temperatures T1 and T2 shown in the display are dependent on the
room
temperature.)
ªA “?”:
comments or tips about the current program step
ªB “Elpho”:
commands to load and start the program
ªC “programs”: commands to edit new programs, change or delete existing programs
ªD “+”:
different options like printing of program stores or
of running protocols, choice of language, choice of
signal
In general select by scrolling, activate by pressing
enter; except when selecting program numbers or
temperature values!
Function key D: Options
1 print programs
2 signal
3 language
A ↑ B ↓ C quit D enter
4 standard mode
5 test mode
6 void
A ↑ B ↓ C quit D enter
ªA ↑ and ªB ↓ allow
scrolling of display.
By ªC “quit” you will return to
the main menu.
1: Printing of program stores. A dot matrix printer can be connected
to the controller by using the port at the rear (see figure 10).
2: Choice whether a beep signal can be heard at the end of a
program or when the program has reached an infinite time step
3: Choice between ªA “German” or ªB “English”
4: not occupied
5: not occupied
6: not occupied
TGGE System
19
Function key C: Editing of programs
program no: __
A list B del C quit
D enter
After entering a number of a
non occupied program store,
the display shows:
Name: >
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ – () α σ /, <>& +. %
A → B ABC C quit D enter
1:L1:__
L6:
alternative:
T1:
T2:
A ? B T1 C quit D →
The temperature gradient
between T1 and T2 must
not exceed 45 K.
In the main menu pressing ªC will offer the possibility to edit a
new program. First choose a program store number.
ªA “list” displays all program stores from 0 to 99 with names or
the information <empty>. ªB “del” deletes the last entry.
To give the program an individual name strike ªB “ABC” to
jump with the cursor into the letter field. Moving inside this field is
possible by the keys ªA “→” and ªB “←”. Pressing ªD “enter”
the current high lighted letter is stored in the name field. This step
can be repeated 8x times. Pressing ªD “enter” two times leads to
the next step.
It is now possible to enter for the first program step temperature
values for both sides of the gradient block:
Alternatively the temperatures for L1 and L6 (first and last thick
lane on the gradient block) or for T1 and T2 (left and right edge of
the gradient block) can be programmed. The change between
programming L and T can be done by pressing ªB "T1". If a
number has been entered at the field L1, you have to confirm by
pressing ªD “enter”. The cursor jumps to L6. After programming
L6 you will be asked "ok ?": Pressing ªB "no→L1" allows new
programming of L1 and L2.Pressing ªD "yes" confirms the
temperatures.
1:L1: 25.0°C
L6:
Alternative:
T1: __
T2:
A ? B delete C quit D →
After entering a temperature for T1, T2, L1 or L6 the temperature
can be Deleted by pressing ªB "delete".
1:L1:
L6:
Alternative:
T1: __
T2:
A ? B L1 C quit D →
If programming of T1 and T2 (left and right edge of the gradient
block) has been done but L1 and L6 are preferred, changing to L1
and L6 can be done by pressing ªB "L1".
1: L1: 25.0°C
L6: 60.0°C
T1: 21.1°C
T2: 63.8°C
Ok ?:
A ? B no→L1 C quit D →
Or
After entering L6 or T2 all four temperatures (L1, L6, T1, T2) are
displayed.
"ok ?": Pressing ªD "yes" leads to the programming of the
Electrophoresis parameters.
Pressing ªB "no→L1" leads back to programming L1. Pressing
ªB
"T1" allows programming of T1 and T2.
Pressing ªB "no→T1" leads back to programming T1. Pressing
ªB "L1" allows programming of L1 and L6.
1: L1: 25.0°C
L6: 60.0°C
T1: 21.1°C
T2: 63.8°C
Ok ?:
A ? B no→T1 C quit D →
Depending on the decision of programming L1 and L6 or T1 and
T2 the programmed temperatures will be shown in the display.
After programming L1 and L6 this temperatures will be shown in
the display during the next programming steps.
TGGE System
1:L1: 25.0°C
L6: 60.0°C
time: __0m 0s
El:
0V
500mA
30W
A ? B V*h
C quit D →
1:L1: 25.0°C
L6: 60.0°C
time: 30m 0s
El:
0V
500mA
30W
A ? B V*h
C quit
D→
1:L1:__
L6:
V*h: __0.00 Vh
El:
0V
500mA
A ? B Time C quit
20
Time: You can choose electrophoresis time. Standard values for a
TGGE run are 30 – 45 min. If you enter 30 and confirm by press
ing ªD “enter” you get 30 s. If you enter 30 x you will get 30 min. If
you enter 30 xx you will get 30 h.
V*h: Pressing ªB "V*h" replaces times by the more precise Volt x
hour integration.
Pressing ªB "Time" replaces V*H by time.
30W
D→
1:L1: 25.0°C
L6: 60.0°C
time: 30m 0s
El: __ 0V
500mA
30W
A ? B special C quit D →
El: Three different electrophoresis parameters (voltage, current or
wattage) can be set. Current and wattage are pre-set at max.
values of 500 mA and 30W respectively. In the beginning we
recommend to set only the Voltage. Depending on the resistance
of the gel electrophoresis the controller will regulate the other two
parameters automatically.
1:L1: 25.0°C
L6: 60.0°C
time: 30m 0s
El: 250V
500mA
30W
A ? B special C quit D →
After confirming the voltage by pressing ªD “enter” and pressing
two times ªD “→”, the following two parameters are not changed.
1: special functions
ramptime: __0m 0s
Ramptime = Ramping time
Pressing ªB "special" gives you the choice to choose how fast
the Gradient block is going to the established gradient. Normally
you choose 1s which means maxi ramping speed. In this case
enter “1” and confirm with ªD “enter”
Pressing ªB "standard" results in the standard display.
A ? B standard C quit D →
1:L1: 25.0°C
L6: 60.0°C
Time: 30m 0s
El: 250V
500mA
30W
A ? B special C quit D →
2:L1:__
L2:
Pressing ªD “→” starts the programming of step 2 in this
program.
alternative:
T1:
T2:
A ? B T1
C quit
D→
program no: ......
pgm end:
.......step(s)
runtime:
....h.....m.......s
By pressing ªC “quit” any change will be saved and the following
messages appear:
L1: 22.0°C
block off
After a few seconds the main menu appears.
L6: 22.0°C
A? B Elpho C programs D +
TGGE System
21
Function key B: Start/Stop
Start block function and electrophoresis
Start program: __
In the main menu pressing key ªB “block” offers the possibility to
start a program.
A list B del C quit
L1: 25.0 °C
D enter
L6: 60.0°C
ramp: 1
time: 0m 1s
El: 250 V 20mA
20W
A list B block C program D +
L1: 25.0 °C
L6: 60.0°C
hold: 1 0m 1s
0.00 Vh
El: 250 V
8mA
20W
A list B block C program D +
Pressing ªC “program” during
block run
Program no:
4
Pgm is active!
A copy B del C quit D display
program no:
4
name: ................
A copy
B
C quit
D display
After entering a program number or choosing a program from the
list
(ªA “list” ) the block starts to establish the gradient. The timer for
the ramping starts immediately. The electrophoresis is started as
soon as the gradient block reaches the programmed temperature
(gradient. The limiting factor (const. V, mA or W) is indicated by an
blinking arrow.)
After establishing the gradient, line two of the display changes.
The timer now starts again and counts the electrophoresis running
time. Additionally the volt/hour integrator starts to count.
It is possible to review a program during run. After pressing ªC
“program” and the corresponding store number a warning
message appears.
Then it is possible to display the active program (ªD “display”) or
to copy it into a new store (ªA “copy”). It’ s not possible to change
the currently running program
By pressing ªC “quit” the main menu appears again.
Stop block function and electrophoresis
program: 0 TEST
To stop a current running program you press ªB “Elpho” and
pause?
again ªD“stop".
stop?
By pressing ªC “quit” you return to previous display without any.
A ? B pause C quit D stop
changes.
When pressing ªD “stop” the run will be aborted and you leave
the actual running program.
Pressing ª B “pause” holds the actual situation of the gradient
L1: 25.0 °C
L6: 60.0°C
hold: 1
pause
0.00 Vh
(stopping electrophoresis, holding the temperature gradient)
El: 250 V
8mA
20W
“pause” is blinking and shown in the display alternatively with the
A ? B Elpho C programs D +
time.
program: 0 TEST
continue?
stop?
A?
B contin
C quit
stop
Pressing ªB "Elpho" (in pause status):
Pressing ªB "contin" will continue the program.
D
TGGE System
22
Function key A "?"
T1: 22.0°C
block off
T2: 22.0°C
Pressing ªA "?" in the main menu results in the following display:
A? B Elpho C programs D +
B: start/stop/pause
C: edit/delete/copy
D. special functions
A? B Elpho C programs D
+
L1: 25.0 °C
L6: 60.0°C
hold: 1 0m 1s
1.16Vh
El: 250 V
8mA
20W
A list B block C program D +
T1: 21.1 °C
L1→3: 21.1
L4→6: 46.0
← = const
T2: 63.8°C
32.0
39.0
53.0
60.0
rtime: h m
Pressing ªA "?" in a running program results in the following display:
The actual temperatures of T1 and T2 as well as the actual temperatures of L1 to L6 are shown in the display.
The limiting factor (const. V, mA or W) is indicated by an blinking
arrow (←).
The actual remaining electrophoresis time is shown on the bottom
(right side of the display).
TGGE System
23
Error messages
TGGE - System
check connection
to thermoblock
TGGE connector cable is not connected to gradient block and / or
system Controller.
Check connections!!!!!
warning:
gradient too large!
max. grad. T1→T2: 45°C
A ? B no→L1 C quit D enter
or
warning:
gradient too large!
max. grad. T1→T2: 45°C
A ? B no→T1 C quit D enter
Programmed temperature gradient too large.
program no:
It is possible to review a program during run. After pressing ªC
“program” and the corresponding store number a warning
message appears.
TEST
pgm is active!
A copy B del C quit D display
program no: __
name:
not programmed!
A ↑ B ↓ C quit
This program number has not been programmed.
D enter
1:L1: __
L6:
entry required
T1:
T2:
A ? B L1
C quit D →
or
1:L1: __
L6:
entry required
T1: __
T2:
A ? B T1
C quit D →
No temperature or time has been programmed.
TGGE System
24
4 Sample preparation
4.1 Purity of samples
Due to the high sensitivity of the staining procedure after TGGE it is recommended to
use purified DNA, RNA or protein samples. Any impurities might be misinterpreted
after TGGE, thereby making the analysis of gels difficult. Nevertheless it is possible
to use even crude mixtures for TGGE analysis.
PCR-amplified DNA fragments can usually be analyzed without purification. But note,
that the presence of high amounts of nonspecific, secondary PCR products may
result in difficulties with interpretation of band pattern, melting profile, etc. For
example, in parallel TGGE, nonspecific bands with a higher molecular weight than
the specific PCR product may be misinterpreted as heteroduplices, or analogs with
lower thermal stabilities. Therefore, before running a TGGE gel, please check the
PCR product and, if necessary, purify the specific PCR product of interest, e.g., by
agarose gel electrophoresis and subsequent gel extraction.
Sample preparation for direct DNA analysis
1 volume of DNA/RNA samples are dissolved with 1 volume of TBE or Na-TAE
loading buffer or with 0.1 volume of the total loading volume ME loading buffer (see
Appendix 2). The resulting mixture is loaded directly on to the polyacrylamide gels.
Secure that the slots are filled up to maximum (if necessary add loading buffer to fill
up the slots to maximum).
In case of low-concentration samples we recommend to prepare 5x conc. loading
buffer. 0.2 volume of this concentrated loading buffer is mixed with the sample and
loaded onto the polyacrylamide gel.
Denaturation/Renaturation for heteroduplex analysis of DNA
For heteroduplex analysis the samples are denatured and renatured prior to TGGE.
Quantitative denaturation is accomplished by heating in 4 M urea. The following
protocol is recommended for all DNA fragments with GC-contents of 50 - 70%.
Depending on the buffer to be used for electrophoresis add one sample volume of
corresponding DR buffer (denaturation/renaturation buffer) to the sample and mix.
Heat at 95°C for 5 minutes (denaturation). Incubate at 50°C for 15 minutes
(renaturation). The sample is then loaded directly to the gel. In order to achieve the
recommended loading volumes for diagonal or perpendicular TGGE (refer to chapter
4.2), the samples should be filled up with running (or loading) buffer.
•
Renaturing at 50°C:
- higher temperatures (higher stringency) can be chosen
for high GC contents to avoid artificial hybrids
- lower temperatures (e.g. 37°C) are applied for expected
hybrids with multiple mismatches or for sequences with
very low GC content. The renaturation temperature should
be approx. 10°C below the Tm of the desired hybrid.
TGGE System
25
4.2 Quantity and volumes of samples
Depending on the slot size of the gel Biometra recommends the following amounts of
material:
50 µl volume,
approx. 50 ng DNA/RNA of interest
5 µl volume,
3 - 5 ng DNA/RNA of interest
glass plate with 1 rectangular slot
+ 2 marker slots
_____________________________________________________________________________________________________
5 µl volume,
3 - 5 ng DNA/RNA of interest
glass plate with 8 slots:
_____________________________________________________________________________________________________
3 µl volume,
1 - 3 ng DNA/RNA of interest
glass plate with 12 slots:
_____________________________________________________________________________________________________
2 µl volume,
approx. 1 ng DNA/RNA of interest
glass plate with 18 slots:
If less volumes fill up the slots with running buffer or loading buffer to the
volumes listed on top! This will create better results.
5 Setting up polyacrylamide gels
5.1 Selecting Concentration of PAA gels
The TGGE System represents a highly optimized system for performing flat bed
polyacrylamide gel electrophoresis under defined temperature conditions. In addition
to typical TGGE applications the system is ideally suited to run standard fragment
separations without temperature gradient very quickly.
Depending on the molecular weight of the sample we recommend the following
acrylamide/bisacrylamide concentrations:
Conc.
3%
5%
8%
DNA fragment length
> 1000 bp
500 - 1000 bp
< 500 pb
5.2 Setting up the gel solution
Each gel sandwich contains approx. 2.5 ml polyacrylamide solution.
We therefore recommend to prepare 10 ml solution to pour 3-4 gels at the same
time. Polymerized gels which are not immediately used must be stored at room
temperature. To inhibit any gel drying we recommend to wrap the polymerized gels
into saran foil or wet plastic bags. Wet towels can be used only for short time
storage.
TGGE System
26
Keep in mind that polymerized polyacrylamide gels which include urea should not be
used after 2 – 4 days of storage (depending on storage conditions)!
27
TGGE System
Recipe for 10 ml gel solution (3 – 4 gels) for TBE running buffer:
Urea (cEnd= 7 M)
Acrylamide/bis Acrylamide
stock solution (30 : 0,8), 40% (w/v)
10x conc.TBE
(cEnd= 0.1 x conc.)
50% Glycerol (cEnd= 2%)
Water, distilled
3% Gel
4.2 g
0.75 ml
5% Gel
4.2g
1.25 ml
8% Gel
4.2 g
2.0 ml
0.1 ml
0.1 ml
0.1 ml
0.5 ml
3.5 ml
0.5 ml
3 ml
0.5 ml
2.5 ml
Make sure that the urea has been completely resolved.
It is possible to heat up the urea containing solution slightly (40°C – 50°C) for a
short time in order to improve the solubilization of urea.
De-gas the solution under gentle vacuum for 3 - 5 min.
Water, distilled
TEMED
APS (4%)
22.5 µl
42 µl
fill up to 10 ml
22.5 µl
22.5 µl
42 µl
42 µl
Mix gently. Avoid air bubbles!
Pour the gel solution into the glass plate sandwich immediately thereafter (see
chapter 4.1.2) without air bubbles.
Recipe for 10 ml gel solution (3 – 4 gels) for Na-TAE running buffer:
Urea (cEnd= 8 M)
Acrylamide/bis Acrylamide
stock solution (30 : 0,8), 40% (w/v)
10x conc. Na-TAE, pH 8.4
(cEnd= 0.2 x conc.)
40% Glycerol (cEnd= 2%)
Water, distilled
3% Gel
4.8 g
0.75 ml
5% Gel
4.8g
1.25 ml
8% Gel
4.8 g
2.0 ml
0.2 ml
0.2 ml
0.2 ml
0.5 ml
3.5 ml
0.5 ml
3.0 ml
0.5 ml
2.5 ml
Make sure that the urea has been completely resolved.
It is possible to heat up the urea containing solution slightly (40°C – 50°C) for a
short time in order to improve the solubilization of urea.
De-gas the solution under gentle vacuum for 3 - 5 min.
Water, distilled
TEMED
APS (4%)
14 µl
45 µl
fill up to 10 ml
14 µl
14 µl
45 µl
45 µl
Mix gently. Avoid air bubbles!
Pour the gel solution into the glass plate sandwich immediately thereafter (see
chapter 4.1.2) without air bubbles.
28
TGGE System
Recipe for 10 ml gel solution (3 – 4 gels) for ME (MOPS/EDTA) running
buffer:
Urea (cEnd= 8 M)
Acrylamide/bis Acrylamide
stock solution (30 : 0,8), 40% (w/v)
50x conc. ME-buffer
(cEnd= 1 x conc.)
40% Glycerol (cEnd= 2%)
Water, distilled
3% Gel
4.8 g
0.75 ml
5% Gel
4.8g
1.25 ml
8% Gel
4.8 g
2.0 ml
0.2 ml
0.2 ml
0.2 ml
0.5 ml
3.5 ml
0.5 ml
3.0 ml
0.5 ml
2.5 ml
Make sure that the urea has been completely resolved.
It is possible to heat up the urea containing solution slightly (40°C – 50°C) for a
short time in order to improve the solubilization of urea.
De-gas the solution under gentle vacuum for 3 - 5 min.
Water, distilled
TEMED
APS (4%)
17 µl
76 µl
fill up to 10 ml
17 µl
17 µl
76 µl
76 µl
Mix gently. Avoid air bubbles!
Pour the gel solution into the glass plate sandwich immediately thereafter (see
chapter 4.1.2) without air bubbles.
5.3 Some remarks corresponding to standard TGGE conditions
Electrophoresis buffer (running buffer):
• Always membrane filtrate (e.g. 0.45µm pore size) the buffers before use!
• Running buffer: always use the concentration identical with the gel condition
• TBE is the most common used buffer system but the electrophoresis is not as fast
as with Na-TAE buffer. It is possible to add up to 5 mM NaCl if a higher ionic
strength is desired, for reversible melting processes which are required for
parallel TGGE in multiple sample analysis. A higher NaCl concentration should
not be used because it causes an unacceptable high electrical current.
• Na-TAE is the buffer for fastest electrophoresis.
• ME buffer meets all the requirements of a variety of TBE-buffers with different
ionic strengths but is only stable for a very short time. (Stable for about 3 days.
Do not use as the buffer becomes yellow.)
• ME buffer allows Na+ concentrations up to 20 mM which greatly favors "reversible
melting" and still allows short run times for TGGE electrophoresis. Mobile Cl- ions
which slow down the migration velocity of nucleic acids are avoided by using the
sodium salt form of MOPS. Due to their reduced mobility the large MOPS anions
keep the current low.
TGGE System
29
Gel conditions:
• 4 M urea can be used for low GC and high degree of mismatches. This
concentration increases Tm instead of the standard concentration (8 M urea) by
approx. 16 - 20°C using TBE buffer or 8 – 12°C using Na-TAE buffer.
• 10 M urea can be used for high GC and lowers Tm instead of the standard
concentration (8 M urea) by approx. 8 - 10°C using TBE buffer or 4 – 6°C using
Na-TAE buffer.
• Glycerol reduces the steepness of very cooperative transition curves, broadening
the profile and expanding the temperature range for detecting small Tm
differences of closely related nucleic acids:
0% Glycerol increases cooperativity (>200 bp, narrower transitions)
>2% Glycerol lowers cooperativity (>200 bp, broader transitions)
TGGE conditions:
• Voltage can be raised to 400 V if the current is below 30 mA
• Current should not exeed 30 mA
Running buffer:
pre-run
run
0.2 x conc. Na-TAE
250 V, 10-12 mA, 2-5 min.
250V, 15-20 mA, 30-60 min.
0.1 x conc. TBE
250 V, 4- 5 mA, 2-5 min.
250 V, 9-12 mA, 30-60 min.
• T1 = 20°C: can be raised to obtain optimized resolution
• T1 = 20°C: lowering should be avoided by using 4M urea
• T2 = 60°C: higher temperatures should be avoided by using 10 M urea or/and 10
mM MOPS.
• T2 = 60°C: can be lowered to obtain optimized resolution
• T2 = 80°C: maximum temperature, can be used if the gel is carefully protected
against evaporation.
Sample preparation:
• Denaturation / renaturation cycle:
• Renaturing at 50°C: higher temperatures (higher stringency) can be chosen
for high GC contents to avoid artificial hybrids
• Renaturing at 50°C: lower temperatures (e.g. 37°C) are applied for expected
hybrids with multiple mismatches or for sequences with very low GC content.
The renaturation temperature should be approx. 10°C below the Tm of the
desired hybrid.
• Samples should be dissolved in buffers with ionic strength identical to the ionic
strength of the running buffer.
• If samples are dissolved in buffer different to the running buffer, the samples have
to be equilibrated against the running buffer (e.g. using dialysis). Nucleic acids
can be precipitated with ethanol and dissolved in denaturation/renaturation buffer
or running buffer.
TGGE System
30
5.4 Assembling the gel sandwich
The thinness of the gel makes it necessary to cast polyacrylamide gels on a gel support
film (Polybond, Order Number: 024-030). Each sandwich consists of four elements:
Bonding glass plate without spacer
Polybond film
Polyacrylamide gel
Glass plate with fixed spacer and fixed slot former (different types of slots
available).
Glass plates
• Glass plates must be dry and free of any dirt or dust. Biometra recommends to
wear powder-free gloves even during cleaning of glass plates in order to prevent any
skin debris which might interfere with silver staining.
• Do not use strong acidic or basic solutions or organic solvents for cleaning the glass
plates.
• Do not incubate glass plates over night in cleaning solutions.
Pretreatment of glass plate with spacer and slot former
• Glass plate with spacer and slot former must be carefully treated with Acryl-Glide
solution (Order Number 211-319) or a similar hydrophobic solution. Drop about 0.5
ml of solution onto the plate and especially between the slot former. (This
protection layer helps to withdraw the polyacrylamide gel from the sandwich after
polymerization.) Wait 2 – 3 min. and than polish the plate with soft paper to
remove any haze!
• This procedure should be repeated after each run.
• Do not drop Acryl-Glide onto the spacer of the glass plate! This possibly leads to
leakage during polymerization.
• Clean spacers with ethanol before assembling the glass plate sandwich.
• If necessary treat the spacers with a small amounts of silicone grease (to protect
leakage).
Polybond film
• Use only original pre-cut Polybond film which perfectly fits onto the gradient block.
• Biometra recommends to use Polybond film only once. Repeated usage and
especially staining might weaken the strength of the Polybond film.
• The Polybond film has two different sides: one hydrophobic side which repels water
and one hydrophilic side on which a water drop will adhere. You can test the different
sides with a drop of water. (The protection paper is attached to the hydrophobic site.)
• Remove the protecting paper sheet before assembling the sandwich. Handle the
Polybond film only with powder-free gloves.
• The hydrophobic side of the Polybond film must be orientated to the Bonding
glass plate. On the hydrophilic side the polyacrylamide gel will polymerize and stick.
• Press the hydrophobic side of the Polybond film firmly to the Bonding glass plate by
using your thumb, a rubber or a gel casting clip. This will prevent that any
polyacrylamide solution running between the Bonding glass plate and the Polybond
film.
31
TGGE System
Glass plate sandwich
• Assemble the Acryl-Glide treated glass plate with spacer, the Polybond film and
the Bonding glass plate as indicated in figure 11.
n
q
o
➌
Figure 11: Setting up a gel sandwich for PAGE.
Bonding glass plate (n), Polybond film (o) and glass plate with spacer and slot
former (p) are assembled (left panel). The Polybond film is only visible at the
inclined edge of the glass plate with spacer (middle panel). Fasten the clamps (q)
above the spacer to increase the pressure and to ensure a leakage free sandwich.
Figure 12: Pouring the polyacrylamide gel.
Initially 1 ml of polyacrylamide gel solution must be poured. The gel sandwich must
be hold at an angle of 45° when pouring (left panel). The solution must run along
one side of the plate sandwich to avoid air bubbles. The remaining 1.5 ml of
solution is poured into the plate sandwich during which time the plate sandwich is
slowly brought back into a vertical position (right panel).
• Fill up the plate sandwich as high as possible. The gel solution is overlayed with
200µl of Isopropyl Alcohol (2-Propanol, Isopropanol) or Isobutyl Alcohol (2Methyl-1-propanol, Isobutanol) or distilled water to produce a horizontal surface
of the gel.
TGGE System
32
• Polymerization of the gel must be at least for 0.5 h, better for 1 - 1.5 h or optional
over night at room temperature. The sandwich should stand up vertically and must
not be moved during polymerization.
• Gels may be stored up to 4 days at room temperature (wrapped in wet paper
towels in a plastic bag). Do not store at 4°C!
5.5 Disassembling the gel sandwich
• Remove the clamps from the plate sandwich.
• Remove the Bonding glass plate from the sandwich by sliding it smoothly!
The gel polymerized to the Polybond film will adhere to the other glass plate.
• Withdraw the Polybond film with the adhering polyacrylamide gel carefully from the
other glass plate. In the area of the slot former remove the Polybond film very
carefully to avoid any damage to the slots.
• If slots show distortion or wrinkles don’t fill in samples because after
electrophoresis bands in this lane will show distortion as well.
TGGE System
33
6 Electrophoresis with the TGGE System
The electrophoresis unit of the TGGE System has been designed to accommodate all
TGGE and related applications like CTGE, TTGE and SSCP, without cumbersome
changes. It’s easy to switch between perpendicular, parallel or diagonal TGGE (for
adequate accessories see chapter 6.4).
6.1 Electrophoresis conditions
The electrophoresis conditions depend on the
kind of material to be separated, e.g. fragment size differences,
kind of application, e.g. parallel or perpendicular TGGE,
sample preparation, e.g. high salt or low salt preparation,
buffer system.
Any recommendations can only be used as guidelines to start with. Further
improvements to the analysis is easily possible by adjusting the run conditions to the
individual needs.
Voltage:
Current:
Run Time:
100 V - 400 V;
5 mA - 25 mA;
10 min - 2 h;
usually 250 V
usually 10-20 mA
usually 30 min
6.2 Preparing the electrophoresis unit
•
•
•
•
Use the leveling eye on the electrophoresis unit and the 4 leveling feet to adjust the
unit.
Remove the safety lid and fill in max. 250 ml of the desired running buffer per
buffer chamber (e.g. 0.1 x conc. TBE, see Appendix 13.2.). The same running buffer
should only be used once.
Soak the pre-cut electrode wicks (order number: 024-020) in the running buffer
before use.
Drop 0.3 - 0.5 ml of thermal coupling solution like 0.1% Triton or 0.1% Tween 20 on
the surface of the gradient block (see figure 13 left panel). The thermal coupling
solution will increase the adhesion of the Polybond film with the attached
polyacrylamide gel and therefore supports temperature equilibration between
gradient block and polyacrylamide gel. The whole block must be covered by the
thermal coupling solution layer. No air bubbles must form.
n
o
➌
Figure 13: Positioning of polyacrylamide gel on gradient block. A small volume of thermal
coupling solution (o) is applied to the gradient block (n)(left panel). The Polybond film with the
polyacrylamide gel on top (➌) is put on the gradient block. Slightly bend the Polybond film (right
panel) in order to spread the thermal coupling solution evenly.
TGGE System
•
•
•
34
To position the polyacrylamide gel on the gradient block the Polybond film should be
held between thumb and middle finger and slightly bended. This leads to an even
distribution of thermal coupling solution beneath the Polybond film.
If air bubbles are visible beneath the polyacrylamide gel, try to squeeze them out
by moving the gel slightly back and forth. If this will not succeed completely remove
the gel and repeat the aforementioned steps. Don’t touch the polyacrylamide gel
directly with your fingers or your gloves.
Excess thermal coupling solution must be removed from the gradient block by
using paper towels.
35
TGGE System
6.3 Perpendicular TGGE
During perpendicular TGGE a mixture of molecules is separated over a wide
temperature range. The temperature gradient is perpendicular to the electrophoresis
run direction.
Steps before TGGE
sample preparation (refer to chapter 4.1)
programming of temperature gradient and electrophoresis parameters (refer to
chapter 3.2.2)
Prepare in advance:
polyacrylamide gel attached to Polybond film
running buffer (250 ml for each chamber)
pre-cut and pre-soaked electrode wicks
pre-cut cover film
cover glass plate (treated on both sides with Acryl-Glide)
•
Because of the fixed orientation of the temperature gradient the removable
electrophoresis chambers must be positioned as indicated in figure 14.
n Platform surrounding the gradient block
o Electrophoresis buffer chamber with
connector for cathode
➌ Gradient block
T1
L1
L2
L
3
L
4
L
5
L
6
T2
q Electrophoresis buffer chamber with
connector for anode
Figure 14: Orientation of the two electrophoresis buffer chambers for perpendicular
TGGE.
•
Fill in the running buffer (e.g. 0.1 x conc. TBE) into each electrophoresis chamber.
Before you place the gel onto the gradient block be sure that the
sample is ready for loading and cover film is available.
•
To make full use of the linear range of the gradient block the polyacrylamide gel
attached to the Polybond film should be positioned as indicated in figure 15. The
rectangular and the marker slots of the gel (p) are positioned at the beginning of the
gradient block (the marked lines represent the beginning of the linear range of the
gradient block).
36
TGGE System
n
➌
➌
o
T1T1
T2T2
T1 T1
TT
22
Figure 15: Orientation of the polyacrylamide gel attached to the Polybond film (n) on
the gradient block (o) during perpendicular TGGE. See the position of the gel slots
(➌) relative to the marked lines of the gradient block.
•
Two pre-cut and pre-soaked electrode wicks must be positioned at the start
and the end of the polyacrylamide gel. Wick and gel have to overlap (see figure
16).
➎
2 - 3 mm
➍
➋
➊
Figure 16: Side view of the polyacrylamide gel (➋) on top of the gradient block during
pre-run. Pay attention to the position of electrode wicks (➍) on top of the
polyacrylamide gel. (Polybond film (➊), slot of polyacrylamide gel (➎)).
•
•
Avoid any contact between sample slots and electrode wicks. Otherwise the
samples will diffuse into the electrode wicks.
Load the samples quickly at room temperature without air bubbles. Do not start
the temperature gradient (the temperature gradient is established after the
samples have fully entered the polyacrylamide gel).
glass plate with 1 rectangular slot
+ 2 marker slots
50 µl volume,
approx. 50 ng DNA/RNA of interest
5 µl volume,
3 – 5 ng DNA/RNA of interest
If less volumes fill up the slots with running buffer or loading
buffer to the volumes listed on top! This will create better results.
37
TGGE System
The time between mounting the gel onto the gradient block and
loading the sample must not exceed 5 minutes.
•
Close the safety lid of the electrophoresis chamber and start electrophoresis at
20°C or 25°C and 250 V for 2 - 5 min. Standard electrophoresis conditions are
given in chapters 5.3 and 6.1.
•
Make sure that the orientation of the gel and the safety lid is exact as indicated in
the following:
T1
•
•
•
•
•
L1
L2
L3
L4
L5
L6
T2
Wait at the electrophoresis chamber until the samples have fully entered the
polyacrylamide gel (unlike with the former TGGE System of QIAGEN this process
will only take 1-3 minutes) and have moved about 3 - 5 mm in the gel.
Stop the electrophoresis run, open the safety lid.
Rinse the - now empty - slots with 0.5 - 1 ml running buffer.
Cover the polyacrylamide gel including the slots with the 7 x 6 cm pre-cut cover
film (see figure 17). A small buffer layer must remain between cover foil and gel.
Avoid air bubbles!
The cover film must be positioned with the long side parallel to the
buffer chambers (= perpendicular to the arrow on the safety lid).
Soak any excessive buffer from the side of the gel. The gel must not swim in
buffer solution.
q
➎
➌
➋
➊
Figure 17: The polyacrylamide gel (o) has to be covered by a pre-cut hydrophobic
cover film (➌). A small buffer layer remains between gel and cover film. (Polybond
film (➊), wicks (q) , slot of polyacrylamide gel (➎))
38
TGGE System
•
•
Bring the electrode wicks to an overlap with the cover film. The overlap
between wick and cover film should be almost 2 cm (see figure 17). Avoid air
bubbles.
Be sure that the 2 silicone barriers are fixed to the cover glass plate before use:
•
Cover the sandwich with the Acryl-Glide treated cover glass plate (see
figure 18).
The silicone barriers have to be positioned perpendicular to the
wicks and never on top of the wicks!
q
➏
➎
➌
➋
➊
Figure 18: Side view of the polyacrylamide gel (➋) on top of the gradient block.
Sandwich of polyacrylamide gel ➋), pre-cut cover foil (➌), electrode wicks (➍) and
cover glass plate with silicone barriers (➏) during perpendicular TGGE run (Polybond
film(➊), slot of polyacrylamide gel (➎)).
•
•
Start the temperature gradient and wait until the gradient has been established
(usually 0.5 - 1 minute).
Start the electrophoresis run.
•
The Bromophenol blue dye only gives you an indication how far the samples have
migrated until you have optimized the best run time.
•
After the electrophoresis run, switch off the controller, open the safety lid of the
electrophoresis unit, remove the polyacrylamide gel and proceed further for
staining the gel (chapter 6.5). It is recommended to fix the gel immediately in
order to improve the analysis.
39
TGGE System
6.4 Parallel TGGE
During parallel TGGE a mixture of molecules is separated over a narrow
temperature range determined by perpendicular TGGE. The temperature gradient is
parallel to the electrophoresis run direction.
Steps before TGGE
sample preparation (refer to chapter 4.1)
programming of temperature gradient and electrophoresis parameters (refer to
chapter 3.2.2)
To prepare in advance:
polyacrylamide gel attached to Polybond film
running buffer (250 ml for each chamber)
pre-cut and pre-soaked electrode wicks
pre-cut cover foil
glass plate (treated on both sides with Acryl-Glide)
•
For parallel TGGE the removable electrophoresis buffer chambers must be
positioned as indicated in figure 19.
n Platform surrounding the gradient
block
o Electrophoresis buffer chamber with
connectors for cathode
T1
L1
L2
L3
L4
L5
L6
T2
➌ Gradient block
q Electrophoresis buffer chamber with
connectors for anode
Figure 19: Orientation of the electrophoresis chambers for parallel TGGE.
•
Fill in the running buffer (e.g. 0.1 x conc. TBE) into each electrophoresis chamber.
Before you place the gel onto the gradient block be sure that the
sample is ready for loading and cover film is available.
•
The polyacrylamide gel attached to the Polybond film must be positioned as
indicated in figure 20. The slots of the gel (p) should be positioned at the
beginning of the gradient block. The first marked line (L1) represents the
beginning of the linear range of the gradient block.
40
TGGE System
n
➌
o
T1
T2
T1
T2
Figure 20: Orientation of the polyacrylamide gel attached to the Polybond film (n) on
the gradient block (o) during parallel TGGE. See the position of the gel slots (➌)
relative to the marked lines of the gradient block.
•
Two pre-cut and pre-soaked electrode wicks must be positioned at the start
and the end of the polyacrylamide gel. Wick and gel have to overlap (see figure
16).
➍
2 - 3 mm
➌
➋
➊
Figure 16: Side view of the polyacrylamide gel (➋) on top of the gradient block during
pre-run. Pay attention to the position of electrode wicks (➍) on top of the
polyacrylamide gel. (Polybond film (➊), pre-cut Polybond film(➌), slot of
polyacrylamide gel (➎)).
•
•
Avoid any contact between sample slots and electrode wicks. Otherwise the
samples will diffuse into the electrode wicks.
Load the samples quickly at room temperature without air bubbles. Do not start
the temperature gradient (the temperature gradient is established after the
samples have fully entered the polyacrylamide gel).
TGGE System
41
Depending on the slot size of the used gel Biometra recommends the following
amounts of material:
5 µl volume,
3 - 5 ng DNA/RNA of interest
glass plate with 8 slots:
_____________________________________________________________________________________________________
3 µl volume,
1 - 3 ng DNA/RNA of interest
_________________________________________________________________________________
glass plate with 18 slots:
2 µl volume,
approx. 1 ng DNA/RNA of interest
glass plate with 12 slots:
If less volumes fill up the slots with running buffer or loading
buffer to the volumes listed on top! This will create better results.
The time between mounting the gel onto the gradient block and
loading the sample must not exceed 5 minutes.
Close the safety lid of the electrophoresis chamber and start electrophoresis at
20°C or 25°C and 250 V for 2 - 5 min. Standard electrophoresis conditions are
given in chapters 5.3 and 6.1.
•
Make sure that the orientation of the gel and the safety lid is exact as indicated in
the following:
•
Wait at the electrophoresis chamber until the samples have fully entered the
polyacrylamide gel (unlike with the former TGGE System of QIAGEN this process
will only take 1-3 minutes) and have moved about 3 - 5 mm in the gel.
Stop the electrophoresis run, open the safety lid.
Rinse the - now empty - slots with 0.5 - 1 ml running buffer.
Cover the polyacrylamide gel including the slots with the 7 x 6 cm pre-cut cover
film (see figure 17). A small buffer layer must remain between cover foil and gel.
Avoid air bubbles!
The cover film must be positioned with the long side parallel to the
buffer chambers (= perpendicular to the arrow on the safety lid).
T1
•
L1
L2
L3
L4
L5
L6
T2
•
•
•
•
Soak any excessive buffer from the side of the gel. The gel must not swim in
buffer solution.
42
TGGE System
q
➎
➌
➋
➊
Figure 17: The polyacrylamide gel (o) has to be covered by a pre-cut hydrophobic
cover film (p). A small buffer layer remains between gel and cover film. (Polybond
film (➊), wicks (q) , slot of polyacrylamide gel (➎))
•
•
Bring the electrode wicks to an overlap with the cover film. The overlap
between wick and cover film should be almost 2 cm (see figure 17). Avoid air
bubbles.
Be sure that the 2 silicone barriers are fixed to the cover glass plate before use:
•
Cover the sandwich with the Acryl-Glide treated cover glass plate (see
figure 18).
The silicone barriers have to be positioned perpendicular to the
wicks and never on top of the wicks!
q
➏
➎
➌
➋
➊
Figure 18: Side view of the polyacrylamide gel (➋) on top of the gradient block.
Sandwich of polyacrylamide gel ➋), pre-cut cover foil (➌), electrode wicks (➍) and
cover glass plate with silicone barriers (➏) during perpendicular TGGE run (Polybond
film(➊), slot of polyacrylamide gel (➎)).
TGGE System
•
•
43
Start the temperature gradient and wait until the gradient has been established
(usually 0.5 - 1 minute).
Start the electrophoresis run.
•
The Bromophenol blue dye only gives you an indication how far the samples have
migrated until you have optimized the best run time.
•
After the electrophoresis run, switch off the controller, open the safety lid of the
electrophoresis unit, remove the polyacrylamide gel and proceed further for
staining the gel (chapter 6.5). It is recommended to fix the gel immediately in
order to improve the analysis.
TGGE System
44
6.5 Silver staining
Aside from autoradiography silver staining is the most sensitive method for detecting
small amounts of DNA, RNA or proteins in polyacrylamide gels. Due to the low
thickness of the gels (0.5 mm) the staining procedure takes no more than 35
minutes.
Other staining protocols may be used, but generally exhibit less sensitivity. This must
be considered in relation to the amount of DNA loaded on the gel.
All incubation steps are done in small plastic containers which are agitated on a
rocking platform (e.g. order number 042-400 or 042-500).
For handling several polyacrylamide gels simultaneously, Biometra offers a semiautomated instrument called Blot Processor (order number 015-000 or 015-090).
Please contact Biometra or your local distributor to receive further information about
the Blot Processor.
Wear non-powdered protective gloves during all steps of the silver
staining protocol to avoid staining artifacts due to the high
sensitivity of the staining protocol.
•
Remove the protective plastic sheets from the gel.
•
Carefully remove any residual thermal coupling solution from the back of the gel
(Polybond film) prior to staining
•
Put the polyacrylamide gel with the gel side upwards into the staining tray. Avoid
air bubbles during all staining steps.
•
It’s recommended to prepare at least 100 ml solution for each incubation step.
•
If NaCl has been added to the gel running buffer, incubate the TGGE gel for 15
min in Fixation solution to remove the NaCl.
45
TGGE System
Standard method:
Step
Fixation
Silver Binding
Washing
Time
5 min
10 min
3 x 1 min
Solutions*
Fixation solution
AgNO3-Solution
Fresh ddH2O
Developing
Stopping
Washing
10 min
5 min
10 min
Developer
Stopping Solution
Rinse under fresh ddH2O
Preparing
storage
for 1-5 h
50% glycerol
Notes
prepare freshly
demineralised
water may be
ok
prepare freshly
Demineralised
water may be
ok
Not absolutely
necessary!
Preparing the gel for storage ( 1 - 5 h at room temperature in 50% glycerol) is not
necessary.
Staining solutions:
Fixation
10%
EtOH
0.5%
Acetic Acid
100 ml ethanol and 5 ml acetic acid are adjusted with distilled water to
1 liter.
Silver Binding
0.19% AgNO3
1.9g AgNO3 is dissolved in 1 liter of distilled water. (Can be reused 5
times)
Store dark!
Developing Solution
1.5%
NaOH
0.08% NaBH4
0.1%
Formaldehyde
Dissolve 15 g NaOH in 1 liter distilled water. Add 0.8g NaBH4 and 2.7
ml formaldehyde stock solution (37% in water).
This buffer must be freshly prepared immediately before use!
Stopping Solution
0.75% Na2CO3
Dissolve 7.5 g sodium carbonate in ddH2O. Total volume: 1 liter
46
TGGE System
Quick method (for PCR products) (Sanguinetti et al):
Step
Fixation
Silver Binding
Washing
Time
3 min
5 min
3 x 1 min
Solutions*
Fixation solution
AgNO3-Solution
Fresh ddH2O
Developing
Stopping
5 min
5 min
Washing
10 min
Developing solution
Ethanol and acetic acid
solution
Rinse under fresh ddH2O
Demineralised
water may be
ok
50% glycerol
Not absolutely
necessary
Room temperature
Preparing
storage
Drying
for 1-5 h
Notes
prepare freshly
prepare freshly
Demineralised
water may be
ok
prepare freshly
Preparing the gel for storage ( 1 - 5 h at room temperature in 50% glycerol) is not
necessary.
Staining solutions:
Fixation:
10%
EtOH
0.5%
Glacial Acid
100 ml ethanol and 5 ml acetic acid are adjusted with double distilled
water to 1 liter. Prepare freshly !
Silver Binding
0.2% AgNO3
2.0 g AgNO3 is dissolved in 1 liter of distilled water. (Can be reused 5
times)
Store dark!
Developing Solution
3.0 % NaOH
0.5%
Formaldehyde
Dissolve 3 g NaOH and 1.35 ml formaldehyde stock solution (37% in
water) in 100 ml double distilled water.
This buffer must be freshly prepared immediately before use!
Stopping Solution:
identical with Fixation solution (10% EtOH, 0.5% Glacial Acid)
47
TGGE System
Quick method using the AMRESCO SilverPAGE staining kit
(Code No. 211-761)
Step
Fixation
Sensibilisation
Washing
Time
15 min
10 min
10 min
Solutions
2 x 100 ml Fixation solution
2 x 100 ml 30% ethanol
3 x 200 ml fresh ddH2O
Silver Binding 15 min
reconstituted Silver Binding
Agent + Formaldehyde
Rinse under fresh ddH2O
Washing
Developing
Stopping
Preparing
storage
Drying
0.5-1
min
1 - 2 min reconstituted Developing
solution + Formaldehyde
5 min
for 1-5 h
7.5% Acetic acid
50% glycerol
Notes
prepare freshly!
Demineralised
water may be
ok
prepare freshly!
!!!!!
prepare freshly!
Develop to
desired level!
Not absolutely
necessary
Room temperature
Preparing the gel for storage ( 1 - 5 h at room temperature in 50% glycerol) is not
necessary.
Staining solutions:
Fixation:
30%
EtOH
10%
Acetic Acid
300 ml ethanol and 100 ml acetic acid are adjusted with double
distilled water to 1 liter.
Sensibilisation:
30%
EtOH
Prepare freshly 60 ml ethanol in 140 ml double distilled water.
Silver Binding:
Prepare Silver Binding Agent by reconstituting contents of one pouch
in 1 l of ddH20. (This solution must be prepared fresh every time!)
Immediately before staining, add 0.7 ml of 37% Formaldehyde to
200 ml of reconstituted Silver Binding Agent.
Developing Solution:
Just prior to use, prepare developing solution by reconstituting
contents of one pouch of Developer I and 15 mg of Developer II in 200
ml of ddH20. (This solution must be prepared fresh every time!)
Immediately before developing, add 0.7 ml of 37% Formaldehyde to
200 ml of reconstituted developing solution.
Stopping Solution:
7.5% Acetic Acid
75 ml acetic acid are adjusted with double distilled water to 1 l.
TGGE System
48
6.6 Ethidium bromide-staining
Incubate the gel in staining solution (0.5 µg/ml ethidium bromide in 1 x conc. TBE) for 30 45 min. Analyze under UV radiation (27).
6.7 Blotting
DNA from TGGE gels can be blotted onto a solid-state support either by electroblotting
(Fastblot) or vacuum-blotting (Vacu-Blot System). If DNA is to be blotted after TGGE
analysis, the TGGE gel must be poured onto the hydrophobic side of the gel support film
(Polybond film). Otherwise, the gel cannot be detached from the gel support film!
6.8 Autoradiography
TGGE gels can also be directly exposed to x-ray films is radiolabeled samples are analyzed.
Direct exposure:
Incubate the TGGE gel for 15 min. in Fixation solution (see 6.5 Silver staining). Optional:
Silver stain the gel.
Remove residual buffer from the gel. Expose to an x-ray film at room temperature.
Exposure of dried TGGE gels:
Incubate the TGGE gel for 15 min. in Fixation solution (see 6.5 Silver staining). Optional:
Silver stain the gel.
Incubate the gel in 2 - 5% glycerol for 10 minutes to prevent the gel from cracking. Incubate
an appropriate sheet of cellophane (no Saran wrap!!!!!) in 2 - 5% glycerol. Layer the
cellophane on the gel. Air dry at room temperature for one day or use a geldryer at 50°C for at
least 3h. Exposure to an X-ray film.
6.9 Elution of DNA from the TGGE gel
DNA fragments which have been separated on TGGE, for example, different alleles of one
gene, can be eluted from silver-stained TGGE gel and reamplified by PCR.
Using a Pasteur pipet, punture the gel and extract a µl piece containing the particular DNA
duplex. Incubate in 20 µl TE buffer overnight. Use a 1 µl aliquot for reamplification.
TGGE System
49
7 TGGE in analysis of point mutations in
dsDNA
For analysis of point mutations in dsDNA, an extremely high detection rate of greater
than 95% is routinely achieved when the experiment is carefully planned. The next
two chapters provide information for optimizing detection of base substitutions.
7.1 Theoretical background of a detection rate approximating 100%
for point mutations - calculations with the POLAND program
DNA does not melt by deannealing base pair by base pair from one end to the other,
but by cooperative denaturation of long stretches, called melting domains. The length
of a melting domain is 25 to several hundred base pairs. The midpoint melting
temperature Tm and the length of a melting domain are mainly determined by the
nucleotide sequence of the DNA. The Tm of DNA fragments differing by even small
changes, such as point mutations, can differ by as much as 1.5°C. When
heteroduplices, hybrids of two species of DNA fragments differing in their base
composition, have been formed, the mismatches lower the Tm value significantly.
Thus the heteroduplex analysis is the preferable because of the additional resolution
provides (1, 28).
The principle by which TGGE uses differences in Tm is that the DNA fragments are
electrophoresed through a linear temperature gradient in the polyacrylamide gel.
When the fragments reach the temperature at which the lowest melting domain starts
to melt, they take on a branched, Y-shaped configuration, which slows down mobility
in the TGGE gel matrix. The electrophoretic migration of fragments differing by single
base changes is retarded by branching at different temperatures, thus they are
resolved from one another during temperature gradient electrophoresis.
The denaturing behavior of any DNA fragment can be predicted, if its sequence is
known. For this purpose the POLAND calculation can be used. The POLAND
software is available in the internet:
http://www.biophys.uni-duesseldorf.de/service/polandform.html.
POLAND software predicts location and Tm values of melting domains for dsDNA
and dsRNA as well as their perpendicular TGGE pattern. The ability to predict the
melting behavior of particular DNA fragments enables one to construct DNA
fragments with optimized melting behavior, resulting in a nearly 100% detection rate
for point mutations inside of this fragment.
Since the end of February 1999 the POLAND program is available in two versions.
Using the above internet address allows the user to select between the old POLAND
request form (Standard), the old POLAND expert request form or the new POLAND
request form. The following information is visible on the screen:
50
TGGE System
Poland Server: ANNOUNCEMENT
___________________________________________________________________________________________
The WWW server for prediction of nucleic acid's thermal stability (called Poland server according to the
author of the basic mathematics) will move during the near future to another computer. This is not a
mere relocation of the program that you have used up to now, but the input/output procedure is
completely rewritten for the new server. The new server produces better/nicer (?) plots and has a
better/more elongated help file. But be aware of new bugs, which might be introduced during the
rewriting and relocation.
The old server, both the standard and the expert form, are unchanged. Both forms will be available for
the near future. However, that server is running on our DEC Alpha under OpenVMS, and we run into
more and more trouble to support that machine.
__________________________________________________________________________________
Now, make a decision:
NEW Server
Poland request form
OLD Server
Poland request form
Poland expert request form
__________________________________________________________________________________
Institut für Physikalische Biologie
(Department of Biophysics)
Heinrich Heine-Universität Düsseldorf, Germany
Feb. 26, 1999
G. Steger / M. Labensky / A. Jäger
TGGE System
7.2
51
The “old” Poland program (Old Server)
7.2.1 About the Poland service
The Poland program is an experimental service of the University of Düsseldorf
Biophysics Department, and thus the whole set-up, access and service are subject to
change.
The Poland server will calculate thermal denaturation profiles and temperaturedependent uv absorbance or gel mobility of double stranded RNA or DNA, based on
sequence input and parameter settings in the request form. - Details below !
The program used in these calculations was developed by Gerhard Steger, for
comparing theoretical predictions to experimental data, mainly optical denaturation
profiles, taken at 260 and 280 nm, and TGGE (temperature gradient gel
electrophoresis) experiments. The original version was written in VAX Fortran (VMS),
using the Graphics Kernel System GKS for data presentation.
7.2.2 Program-specific information
Calculation is based on D. Poland's algorithm in the implementation described by
Gerhard Steger.
The Poland algorithm calculates the denaturation profile for double-stranded nucleic
acid using nearest-neighbor stacking interactions and loop entropy functions
described in the literature. An extension of the algorithm, the 'virtual stack' model,
allows for the incorporation of specific mismatched sequence positions in the stability
calculations, as described by Heinz Werntges.
The input data required for calculation are:
• the sequence (≤ 1,000 bp). Use GCG-format or plain format without spacing.
Plain format accepts only 180 characters per line!
• mismatched positions (optional),
• the strand concentration, affecting the dissociation temperature (use the
programmed standard),
• parameter set selection (DNA/DNA low salt/RNA; oligo/long ds),
• output format options (choose GIF format).
Data sets predicted by the program comprise the following:
• A perspective view on the temperature-dependent denaturation profile, that is
denaturation probability vs. sequence position vs. temperature.
• The temperature-dependent relative uv hypochromicities as they would be
measured in optical melting, at wavelengths of 260 and 280 nm (282 nm in
case of DNA), respectively ( full hypochromicity corresponds to approx. 30%
of the OD at low temperature ).
• The derivative form of above hypochromicities, showing the melting
temperature(s) and corresponding half width(s) of the transitions(s), giving
hints about the transition cooperativity.
TGGE System
52
•
Predicted relative gel mobility as calculated according to Lerman et al., as a
graph vs. temperature for different values of the 'retardation length' parameter.
This plot can be used for direct comparison with TGGE experiments;
superpositions of plots generated with or without mismatched positions given
are
useful as a hint whether specific mismatched duplexes could be detected
among homoduplexed DNA in a mixture of sample and reference double
strands having undergone a denaturation-renaturation cycle, using either
perpendicular or parallel TGGE.
• A 'half-denaturation temperature' plot showing the half-denaturation
temperature for each base. This plot can also be used to estimate the
destabilizing effect of mismatches on the surrounding part of the sequence: a
temperatureshift of the TGGE transition can be expected if the lowest melting part of the
sequence is directly affected by the mismatch!
Calculations can be done for oligonucleotides (>15 bases) or long double strands
(>50 bases), respectively. In the case of oligonucleotide mode, a length-dependent
correction for the strand dissociation process is applied; the temperature range is
adapted as well. We do not have sufficient experimental results to stringently check
for this mode to give valid results, but for the length range of about 20 nucleotides
there is at least experimental evidence. Using 'oligo' mode with far longer sequences
gives misleading results!
Graphics output is possible in Postscript, HPGL, GIF and PBM format, numeric
results are available as well. All graphics results are directly sent to the WWW client,
for GIF inline images and pbm images links are provided to retrieve a copy for the
external viewer (or for download to disk).
53
TGGE System
7.2.3 How to use the “old” Poland program (Standard)
Poland service request form
This form is an experimental service of the Biophysics Department, further informations are
available here!
The Poland server will calculate the thermal denaturation profile of double stranded RNA or
DNA based on sequence input and parameter settings in this form.
Calculation is based on D. Poland's algorithm in the implemetation described by G. Steger.
Calculations can be done for oligonucleotides (>15 bases) or long double strands (>50 bases),
respectively. A form allowing for 'expert' parameter settings is available, too. Graphics output is
possible in Postscript, HPGL, GIF and PBM format, numeric results are available as well.
Graphics results are directly sent to your WWW client.
For a (more or less) detailed description of the various parameters, you may read a help page.
Sequence title line:
Sequence:
(plain format without spacing; max. 180 chars per line)
Mismatched positions:
(comma-separated)
Strand concentration:
(default 1.0e-6 M; don't give the unit)
Thermodynamic parameters:
Sequence length:
DNA default parameters.
oligonucleotide or
long double strand ?
Output options: __
Click here to
click here to
GIF inline images.
submit
reset
, or
the form to defaults.
If you have comments or suggestions on this service, you can send us mail here !
BiophysWWW / G. Steger Oct. 1996
TGGE System
Working with the web-based POLAND program only need 4 steps:
1. Enter DNA sequence (≤ 1000 bases)
2. Enter mismatch position (optional)
3. Choose GIF format
4. Press submit
54
TGGE System
7.3
55
The “new” Poland program (New Server)
7.3.1 About the Poland service
The Poland server will calculate thermal denaturation profiles and temperaturedependent UV absorbance or gel mobility of double stranded RNA, DNA, or
RNA/DNA-hybrids based on sequence input and parameter settings in the Poland
request form. -- Details of the Poland program are given below.
The program used in these calculations was developed by Gerhard Steger for
comparing theoretical predictions to experimental data, mainly optical denaturation
profiles, taken at 260 and 280 nm, and TGGE (temperature gradient gel
electrophoresis) experiments.
The original version was written in VAX Fortran (VMS), using the Graphics Kernel
System GKS for data presentation.
7.3.2 Program-specific information
Calculation is based on D. Poland's algorithm including the modification by Fixman &
Freire in the implementation described by Gerhard Steger. The Poland algorithm
calculates the denaturation profile for double-stranded nucleic acid using nearestneighbor stacking interactions and loop entropy functions described in the literature.
The input data required for calculation are:
•
•
•
•
•
•
the sequence, of course (and no default here!),
optional mismatched positions,
the strand concentration, affecting the dissociation temperature,
the method to calculate the final dissociation into single strands,
the thermodynamic parameter set (DNA/DNA low salt/RNA), and
the temperature range in which the calculation is performed.
In case you need access to the full range of input options, more options are available
to the experts. Data sets predicted and figures drawn by the program are described
below; see also for OUTPUT.
•
•
•
A perspective view on the temperature-dependent denaturation profile
(denaturation probability vs. sequence position vs. temperature. This plot does
not include the dissociation of dsNA into single-strands; thus it shows most clearly
the relative stability of the different parts of the NA.
The temperature-dependent relative UV hypochromicities as measured in optical
melting, at wavelengths of 260 and 280 nm (282 nm in case of DNA), respectively
(full hypochromicity corresponds to approx. 30% of the OD at low temperature).
The derivative form of above hypochromicities, showing the melting
temperature(s) and corresponding half width(s) of the transitions(s), giving hints
about the transition cooperativity.
TGGE System
•
•
56
Predicted relative gel mobility, calculated according to Lerman et al., vs.
temperature for different values of the 'retardation length' parameter Lr. This plot
can be used for direct comparison with TGGE experiments; superpositions of
plots generated with or without mismatched positions given are useful as a hint
whether specific mismatched duplexes could be detected among homoduplexed
DNA in a mixture of sample and reference double strands having undergone a
denaturation-renaturation cycle, using either perpendicular or parallel TGGE.
A 'half-denaturation temperature' plot showing the temperature at which each
base pair has a probability of 50% to be in the open state. Similar to the threedimensional plot, this plot can be used to estimate the destabilizing effect of
mismatches on the surrounding part of the sequence: a temperature-shift of the
TGGE transition can be expected if the lowest melting part of the sequence is
directly affected by the mismatch.
Calculations can be done for oligonucleotides (>15 bases) or long double strands
(>50 bases), respectively. In the case of oligonucleotide mode, a length-dependent
correction for the strand dissociation process is applied. We do not have sufficient
experimental results to stringently check for this mode to give valid results, but for the
length range of about 20 nucleotides there is at least experimental evidence. Using
'oligo' mode with far longer sequences gives misleading results!
7.3.3 References for Poland Service
Description of implemented programs
Steger, G. (1994). Nucleic Acids Res. 22, 2760-2768.
Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient
gel electrophoresis and polymerase chain reaction.
Original version of algorithm:
Poland, D. (1974). Biopolymers 13, 1859-1871.
Recursion relation generation of probability profiles for specific-sequence macromolecules with longrange correlations.
Fixman & Freire (1977). Biopolymers 16, 2693-2704.
7.3.4 HELP for Poland Service:
7.3.4.1
POLAND
The program POLAND simulates transition curves of double-stranded nucleic acids
(DNA and RNA as well as DNA/RNA hybrids).
Additional information available:
OUTPUT, RELATED PROGRAMS, RESTRICTIONS, ALGORITHM,
SUGGESTIONS, PARAMETERS
TGGE System
7.3.4.2
57
OUTPUT
The program writes it output in numeric format, which is converted to graphics by
Tk/Tcl.
Additional information available:
General description, resolution, 3Dplot, GelPlot, MeltPlot, Temp50%Plot
Resolution of graphics output
The primary graphics output is produced as PostScript® (vector format). That format
is converted to GIF® (raster format); this is a format directly displayed by your WWW
browser. The resolution of the GIF images is selectable: 72 dots per inch (72 dpi) is
the standard screen resolution; 150 dpi or 300 dpi are nice for printing. But be aware
on NanoWeak® systems: the higher resolutions need a lot of memory and tend to
crash your system.
3DPlot
Probability of an open base-pair is plotted as a function of position in sequence and
temperature.
GelPlot
Relative mobility is plotted as a function of temperature for the three different
stiffness parameters.
MeltPlot
Relative hypochromicity and its derivative is plotted as a function of temperature at
260 nm and 282 nm (RNA 280 nm).
References:
for RNA:
Coutts, S.M. (1971). Biochim. Biophys. Acta 232, 94-106. Thermodynamics and kinetics of GC base
pairing in the isolated extra arm of serine-specific tRNA from yeast
for DNA:
Blake, R.D. & Haydock, P.V. (1979). Biopolymers 18, 3089-3109. Effect of sodium ion on the highresolution melting of lambda DNA
Temp50%Plot
Temperature is plotted at which the corresponding base stack has a probability of
50% to be in the open state. The two horizontal lines in the plot mark the temperature
range of calculation; i.e., a curve coinciding with such a line is not valid.
7.3.4.3
RELATED PROGRAMS
The Poland program calculates the denaturation behavior of double-stranded NA.
LinAll, RNAfold, and mFold calculate the secondary structure of single-stranded
(R)NA; in addition LinAll and RNAfold allow the prediction of denaturation behavior of
ssRNA.
TGGE System
7.3.4.4
58
Restrictions
The sequence has to be shorter than 1001 nucleotides but longer than 5 nucleotides.
Valid nucleotides are A, G, C, U, and T.
Calculation of asymmetric or bulge loops is not possible.
7.3.4.5
Algorithm
Calculation is based on Poland's algorithm including the modifications proposed by
Fixman & Freire.
With the original algorithm of Poland computing time is proportional to the square of
the sequence length.
With the modification according to Fixman & Freire computing time is proportional to
10 times the sequence length, but it works only with loop parameters according to
Poland.
7.3.4.6
SUGGESTIONS
Hints for combination of parameters and their values.
Additional information available:
RNA
DNA
RNA/DNA
Ionic_strength_dependence
RNA
Thermodynamic values according to Turner et al. are ideally suited for calculation in
1 M ionic strength after correction of all DeltaS values by 1.021 and all DeltaSGC
values by 0.961. These corrections are equivalent to a shift in Tm values of A:U
stacks by -7 K or -2%, respectively and of G:C/G:C stacks by +7 K or +2%,
respectively.
Optimal (?) parameter combination for Turner et al.:
-d 1.021 1.000 0.961 (DeltaS, DeltaS(A:U), and (DeltaS(G:C) factor)
-n 1.e-3
(Dissociation constant ß)
-c 1.e-6
(ß*c0 = 1E-8 to 1E-10)
-s 1.000
(loop parameter Sigma)
-l g
(internal loops according to Gralla & Crothers)
-t 90. 120. 0.5
(Temperature range and steps)
TGGE System
59
Optimal (?) parameter combination for Pörschke et al.:
-d 1.000 1.040 0.970 (DeltaS, DeltaS(A:U), and (DeltaS(G:C) factor)
-n 1.e-3
(Dissociation constant ß)
-c 1.e-6
(ß*c0 = 1E-9 to 1E-11)
-s 1.e-6
(loop parameter Sigma)
-l p
(internal loops according to Poland)
-a f
(algorithm according to Fixman & Freire)
-t 90. 120. 0.5
(Temperature range and steps)
DNA
Thermodynamic values according to Gotoh et al. and Klump, both, are ideally suited
for calculations. The parameter set of Breslauer et al. does not fit our experiments
(?). The parameter set of Allawi & SantaLucia is based on a reevaluation of all known
parameter sets for DNA; i.e., this set may the optimal one. For references to the
original thermodynamic parameter sets see here.
Additional information available:
Gotoh
Breslauer et al.
Klump
SantaLucia et al.
Allawi & SantaLucia
• Gotoh
Optimal (?) parameter combination for Gotoh:
-d 1.000
-n 1.e-3
-c 1.e-6
-s 1.e-3
-l p
-a f
-t 60. 80. 0.5
(DeltaS factor)
(Dissociation constant ß)
(ß*c0 = 1E-9 to 1E-11)
(loop parameter Sigma)
(internal loops according to Poland)
(algorithm according to Fixman & Freire)
(Temperature range and steps)
• Breslauer et al.
No optimal parameter combination for Breslauer et al.
• Klump
Optimal (?) parameter combination for Klump:
-d 1.000
-n 1.e-3
-c 1.e-6
-s 1.e-3
-l p
-a f
-t 70. 90. 0.5
(DeltaS factor)
(Dissociation constant ß)
(ß*c0 = 1E-9 to 1E-11)
(loop parameter Sigma)
(internal loops according to Poland)
(algorithm according to Fixman & Freire)
(Temperature range and steps)
TGGE System
• SantaLucia et al.
• Allawi & SantaLucia
This is the "unified parameter set"!
RNA/DNA
Thermodynamic values according to Sugimoto et al. (1995) in 1 M NaCl. The top
strand is RNA, the bottom strand is DNA (5'-r-3'/3'-d-5'); the input sequence is the
RNA strand.
Ionic strength dependence
Following values may be used for correction of calculated Tm-values:
Tm,2 - Tm,1
---------- = f(G:C)*I(G:C) + (1-f(G:C))*I(A:U)
log(c2/c1)
with Tm
c
f(G:C)
I(X:Y)
DNA
I(A:T)
I(G:C)
RNA
I(A:U)
I(G:C)
= transition (midpoint, melting) temperature
= ionic strength (=concentration of Na ions)
= G:C content
= dependence of ionic strength of
base pair type X:Y
= 18.3 °C (Owen, Hill, & Lapage (1969).
Biopolymers 7, 503-516.)
= 11.3 °C (Frank-Kamenetskii (1971).
Biopolymers 10, 2623-2624.)
= 20.0 °C (Steger, Müller & Riesner (1980).
= 8.4 °C Biochim. Biophys. Acta 606, 274-284.)
60
TGGE System
7.3.4.7
61
PARAMETERS
Additional information available:
BASE_STACKING_(thermodynamic_parameters)
ENTROPY_CORRECTION_of_base_stacking
LOOP_PARAMETERS_(thermodynamic_parameters)
TEMPERATURE_RANGE_OF_CALCULATION
MISMATCHED_POSITIONS_in_original_sequence
CONCENTRATION_and_DISSOCIATION_CONSTANT
STIFFNESS_of_nucleic_acid
THERMODYNAMIC PARAMETER SETS for BASE STACKING
You can select between five different thermodynamic parameter sets of base
stacking (for loop parameters see below):
Additional information available:
RNA
DNA
RNA/DNA
•
RNA
• for RNA in 1 M NaCl
Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T. & Turner, D.H.
(1986). Proc. Natl. Acad. Sci. USA 83, 9373-9377.
Improved free-energy parameters for predictions of RNA duplex stability.
•
for RNA in 1 M NaCl
Pörschke, D., Uhlenbeck, O.C. & Martin, F.H. (1973). Biopolymers 12, 1313-1335.
Thermodynamics and kinetics of the helix-coil transition of oligomers containing GC base pairs.
•
DNA
• for DNA in 0.019 M NaCl
Gotoh, O. (1983). Adv. Biophys. 16, 1-52.
Prediction of melting profiles and local helix stability for sequenced DNA. •for DNA
•
in 1 M NaCl
Breslauer, K.J., Frank, R., Bloecker, H. & Marky, L.A. (1986). Proc. Natl. Acad. Sci. USA 83, 37463750.
Predicting DNA duplex stability from the base sequence. •
• for DNA in 0.1 M NaCl
Klump, H.H. (1987). Canad. J. Chem. 66, 804-809.
Energetics of order/order transitions in nucleic acids.
TGGE System
62
Klump, H. (1990). in Landolt-Börnstein, New Series, Group VII Biophysics, Vol. 1 Nucleic Acids,
Subvol. c Spectroscopic and Kinetic Data, Physical Data I, (W. Saenger, ed.), Springer-Verlag
Berlin, p. 244-245.
Calorimetric studies on DNAs and RNAs.
•
for DNA in 1 M NaCl
SantaLucia, J. Jr., Allawi, H.T. & Seneviratne, P.A. (1996). Biochemistry 35, 3555-3562.
Improved nearest-neighbor parameters for predicting DNA duplex stability.
for DNA in 1 M NaCl
Allawi, H.T. & SantaLucia, J. Jr. (1997). Biochemistry 36, 10581-10594.
Thermodynamics and NMR of Internal G·T Mismatches in DNA.
•
RNA/DNA
• for RNA/DNA hybrids in 1 M NaCl
Sugimoto, N., Nakano, S., Katoh, M., Matsumura, A., Nakamuta, H., Ohmichi, T., Yoneyama, M. &
Sasaki, M. (1995). Biochemistry 34, 11211-11216.
Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes.
• ENTROPY CORRECTION of base stacking
(Option not available by WWW)
DeltaS values of base stacking may be corrected by factors in order to simulate
deviating ionic strengths.
The Delta S values of the thermodynamic parameters are multiplied with these
factors. The first is used for correction of all Delta S values, the second only for A:U
stacks, the third only for G:C stacks.
Different values are used as defaults in dependence on the chosen thermodynamic
parameter set.
• LOOP PARAMETERS (thermodynamic parameters)
(Option not available by WWW; i.e., Sigma is fixed to 1.e-3, and loop entropy is
calculated according to Poland.)
Loops which appear during denaturation by internal base stack opening may be
calculated by three different methods:
• -l p ==> DeltaS(loop) = SIGMA*(loop+1)**-1.75
(according to Poland or Fixman & Freire)
Use only with stacking parameters according to
Pörschke et al. (-p p),
Gotoh
(-p g), or
Klump
(-p k).
• -l g ==> DeltaS(loop) = SIGMA*DeltaS(loop)
(according to Gralla & Crothers)
Use only with stacking parameters according to
Turner et al.
(-p t).
• -l t ==> DeltaS(loop) = SIGMA*DeltaS(loop)
(according to Turner et al.)
Use only with stacking parameters according to
Turner et al.
(-p t).
TGGE System
63
Therefore, Sigma influences the cooperativity and the half width of each transition.
With '-a f' you can change the default algorithm (only in case '-l p'). With the original
algorithm of Poland (default), computing time is proportional to the square of the
sequence length. With '-a f' the modified algorithm of Fixman & Freire is used which
results in computing time proportional to 10 times the sequence length but works only
with loop parameters according to Poland (-l p) up to a sequence length of 1000 base
pairs.
References:
Poland, D. (1974) Biopolymers 13, 1859-1871. Recursion Relation Generation of Probability Profiles
for Specific-Sequence Macromolecules with Long-Range Correlations.
Fixman and Freire (1977) Biopolymers 16, 2693-2704. Theory of DNA melting curves.
Gralla, J. & Crothers, D.M. (1973) J. Mol. Biol. 78, 301-319. Free energy of imperfect nucleic acid
helices. III. Small internal loops resulting from mismatches.
Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T. & Turner, D.H.
(1986) Proc. Natl. Acad. Sci. USA 83, 9373-9377. Improved free-energy parameters for predictions of
RNA duplex stability.
• TEMPERATURE RANGE OF CALCULATION
The temperature range for calculations has to be adapted to the other parameters;
thus see the topic Suggestions.
In principal not more than 110 temperature points are allowed for a single calculation.
• MISMATCHED POSITIONS in original sequence
Mismatches are given as a comma-separated list of sequence positions; f.e.
-m 2,3,111
specifies mismatched 'base pairs' at positions 2, 3, and 111. If the mismatch is longer
than a 'base pair', the position of each base has to be given separately. The
sequence position of the mismatched base pair(s) may be given in any order.
Calculation of asymmetric or bulge loops is not possible; these have to be modeled
by larger mismatches (internal loops).
• CONCENTRATION and DISSOCIATION CONSTANT
-c 1.e-6 Concentration of single strands C0
-n 1.e-3 Dissociation constant ß
ß*c0 influences temperature Tm and half width of the second order transition, i.e. the
strand separation.
The dissociation constant ß has to be in the range 1. => ß => 1.E-5., and the strand
concentration has to be in the range 1. => C0 => 1.E-13.
If case of short oligonucleotides, ß might be calculated according to
Benight, A.S. & Wartell, R.M. (1983). Biopolymers 22, 1409-1425. Influence of basepair changes and cooperativity parameters on the melting curves of short DNAs.
and
Benight, A.S., Wartell, R.M. & Howell, D.K. (1981). Nature 289, 203-205. Theory
agrees with experimental thermal denaturation of short DNA restriction fragments.
TGGE System
64
• STIFFNESS of nucleic_acid (Lr)
Stiffness of nucleic acid or gel pore size is given f.e. with
-l 40 90 200.
References:
Lerman, L.S., Fischer, S.G., Hurley, I., Silverstein, K. & Lumelsky, N. (1984). Ann. Rev. Biophys.
Bioeng. 13, 399-423.
Fischer, S.G. & Lerman. L.S. (1982). Proc. Natl. Acad. Sci. USA 80, 1579-1583.
Riesner, D., Henco, K. & Steger, G. (1991). In: Advances in Electrophoresis, Vol. 4 (Chrambach, A.,
Dunn, M.J. & Radola, B.J., eds.) VCH Verlagsgesellschaft, Weinheim, pp. 169-250.
Temperatur-gradient gel electrophoresis: A method for the analysis of conformational transitions and
mutations in nucleic acids and proteins
___________________________________________________________________
Institut für Physikalische Biologie
(Department of Biophysics)
Heinrich Heine-Universität Düsseldorf, Germany
Feb. 26, 1999
G. Steger / M. Labensky / A. Jäger
65
TGGE System
7.3.5 How to use the “new” Poland program
Poland service request form
Sequence title line:
Sequence:
(plain format;
no numbers;
max. 1000 nts;
min. 5 nts)
Mismatched positions:
(comma-separated numbers)
Thermodynamic parameters:
Dissociation constant ß:
DNA (100 mM NaCl, Klump).
Oligonucleotide
(ß is function of seq.length)
Long double strand
(default: ß=1.0E-3/M)
Strand concentration:
(default: 1.0E-6 M)
Temperature range:
Which graphics do you want:
Graphics size:
(GIF format)
Click here to
Click here to
submit
Reset
Low temperature limit: High temperature limit: Temperature step
(default: 110.0°C)
size: (default: 2.0°C)
(default: 40.0°C)
Tm(p=50%)
plot
3d
plot
Mobility
plot
Melting
curve
72x72 dpi
, or
the form to defaults.
Institut für Physikalische Biologie
(Department of Biophysics)
Heinrich Heine-Universität Düsseldorf, Germany
Feb. 26, 1999
G. Steger / M. Labensky / A. Jäger
Diff. melting
curve
TGGE System
7.4
66
The optimized DNA fragment
The optimized fragment for detection of point mutations in dsDNA is derived by PCR
amplification and has a length of 200 / 300 to max. 800 / 900 bp. It consists of 1 - 2
melting domains derived from the native sequence plus a synthetic stabilizing region
(GC-clamp). The GC clamp is highly stable because of 3 hydrogen bridges between
G and C whereas there are only 2 hydrogen bridges between A and T. This clamp
may either consist of a 40 bp artificial stretch of GC base pairs (29) or a covalent
chemical clamp (Psoralen = Furo[3,2-g]coumarin, C11H6O3). Psoralen is the better
choice for temperature gradients at high temperatures because Psoralen intercalates
with the double helix and after UV-treatment it links both strands covalently
(irreversible binding). Both clamps are introduced into the DNA fragment by a 5'
overhang of one of the PCR amplification primers. (For easier reading of the
following text both kinds of clamps will re referred to as GC-clamps" further on.)
The melting properties of a DNA fragment are best described by the two-dimensional
"TempPlot diagram" (fig. 21). In this diagram the Tm value is given on the y-axis and
the base pair number on the x-axis. The optimal fragment for TGGE analysis shows
a "stair type" profile with 2 or 3 "steps", respectively (fig. 21, lower diagram). The
highest "step" (the melting domain with the highest melting Temperature Tm) is the
artificial GC-clamp. Length and midpoint melting temperature of the 1 - 2 lower
"steps" (melting domains with lower Tms) are determined by the original sequence of
the DNA under study.
Figure 21: Constructing the optimized DNA fragment
TGGE System
67
When constructing an optimized fragment, start with the "TempPlot" of an
approximately 1000 bp sequence. For PCR amplification, select a fragment
consisting of 1 - 2 melting domains. Put the GC-clamp at the more stable end or at
any end, if the fragment contains one melting domain. Figure 22 schematically
demonstrates how fragments with the utmost longest part of the sequence could be
selected according to the "TempPlot" diagram.
The primers used for PCR amplification have to meet the following rules:
• Use non complementary primer sequences. Do not allow base-pairings of the last
3 bases at the 3'-end, neither with any other bases in the primer itself, nor with the
counterpart primer.
• Select primers 20 - 25 bp in length
• Be sure that there are no additional primer annealing sites in the DNA sequence.
Figure 22: Schematic "TempPlot" diagram of a DNA sequence
TGGE System
68
Fragments which have not been "clamped" may also be analyzed on TGGE, but they
must contain at least two melting domains. In this case, the most stable melting
domain may act as a "natural GC-clamp", provided that electrophoresis is terminated
before this second domain reaches its respective Tm. Under these experimental
conditions nucleotide changes within this highest melting domain will not lead to a
shift of bands on the TGGE gel, and hence, will not be detectable. In conclusion, the
absence of GC-clamps will still allow nearly 100% detection rate for mutations in the
low melting temperature domain(s), but virtually no ability to detect mutations in the
domain with the highest Tm.
7.4.1 Asymmetric GC-clamps for PCR primers used for TGGE
analysis
The 5’ end or the 3’ end of the primer for the end of the segment at which a clamp is
optimal must carry a GC-clamp. The length of the clamp depends on the sequence of
the sample. The denaturing behavior of the modified sample can be tested using the
POLAND software.
short GC-clamp (23 bp):
cccgc cgcgc cccgc cgccc gcc
long GC-clamp (40 bp)44:
cgccc gccgc gcccc gcgcc cggcc cgccg ccccc gcccg
long GC-camp (39 bp)45:
ccccg ccccc gccgc ccccc ccgcg cccgg cgccc ccgc
7.4.2 Chemical clamp with Psoralen (Furo[3,2-g]coumarin, C11H6O3)
The 5’ end of the primer for the end of the segment at which a clamp is optimal
(POLAND program) must carry a appropriately linked psoralen moity at the end
adjacent to T or A, preceding the genomic sequence. The optimal primer sequence
may be 5’(Pso)pTaPpnpnp.....3’, given the preference of psoralen for binding
between TpA and ApT pairs13,46,47. Crosslinking of the PCR product is done e.g. in a
flat-bottom microtiter plate using a 365 nm UV source. Working with small volumes it
may be necessary to minimize evaporation by cross-linking at 4 – 10°C. The yield is
not affected by temperature. The distance of the sample from the UV source affects
the yield. 15 min at 0.5 cm distance of the sample from an 8 W UV lamp is sufficient.
69
TGGE System
7.4.3 POLAND analysis of samples
Unoptimized DNA fragment
Domains not distinctly different
Second order line is flat
Optimized DNA fragment (GC clamp attached)
Domains distinctly different
Two distinct second order line plateaus
Optimized DNA fragment with mismatch
Mismatch position
Mismatch changes melting behavior
TGGE System
70
8 Optimizing parallel TGGE by perpendicular
TGGE
8.1 Check short DNA fragments for their melting behavior
All short DNA fragments (100 - 150 bp) should be checked first by a perpendicular
TGGE gel. This is not only a good place to start for practical optimization of parallel
TGGE, but also verifies the reversible melting behavior of the DNA fragment (fig. 23).
"Reversible melting" can only occur if the DNA fragment consists of at least two
separate melting domains (fig 23 a, c, d). Reversible melting behavior must be
verified since it is required for successful parallel TGGE analysis. Thus, be sure to
check all fragments on a perpendicular TGGE gel which do not contain 40 bp GCclamp or which have not been evaluated with the aid of the POLAND program.
Figure 23: Short DNA fragments without GC-clamp
TGGE System
71
8.2 From perpendicular to parallel TGGE
Perpendicular TGGE routinely uses a standard temperature gradient from 20 - 60°C
in combination with buffers that contain minimum 8 M urea. Using this same standard
gradient in parallel TGGE is possible, but time-consuming, since a longer running
time is required to move the sample from the slot (top of the gel) to the effective
range of separation (middle or lower part of the gel where the domains begin to melt).
In other words, much of the time consumed by electrophoresis is nonproductive since
melting will not begin to occur until the DNA fragment has migrated a considerable
distance.
Parallel TGGE can be easily be optimized by the information acquired from a
preliminary perpendicular TGGE gel.
Figure 24: From perpendicular to parallel TGGE
Steps:
1. Determine the temperature range of effective separation. This temperature
interval is defined by two temperatures, Tlow and Thigh. If possible, use two
different DNA fragments, one wild-type and one mutated, and perform
heteroduplex analysis. In this case, Tlow is determined by the highest temperature
where all duplices remain double-stranded (no retardation in the gel), Thigh is
determined by the melting temperature (Tm) of the most stable homoduplex (see
fig. 24a). In the event that only a wild-type sequence is available, determine Thigh
by the melting temperature Tm of this sequence, and define Tlow = (Thigh - 10°C).
Note: The temperature range of effective separation will have to be determined
for each new DNA fragment analyzed.
2. Using the information from step 1, select a temperature gradient for parallel
TGGE which overlaps the range of effective separation. Program the temperature
of Tlow for L1 (first thick lane on the gradient block, close to T1) and program the
temperature of Thigh + 5°C for L6 (last thick lane on the gradient block, close to T2)
for the parallel TGGE run (fig. 24b).
TGGE System
72
9 TGGE / SSCP
9.1 Running an SSCP on the TGGE
TGGE can be used in combination with SSCP ("single strand conformation
polymorphism") to improve (often dramatically) the frequency of detecting SSCP
markers. TGGE/SSCP is non-radioactive, because it utilizes silver staining detection.
SSCP relies upon the separation of single-stranded DNA or RNA which have formed
hairpin secondary structures. Different conformations exhibit different electrophoretic
mobilities. The conformation which a particular single-stranded molecule adopts is
sequence-dependent, and mutations are detected by their influence upon the
secondary structure, and hence, the altered electrophoretic mobility.
Figure 25: Effect of conformational differences
As indicated in figure 25, conformational differences between two different basesubstituted fragments can be achieved only within a limited temperature range (Tlow T high): At temperatures below Tlow, both fragments adopt the characteristic hairpin
structure. On the other hand, at temperatures higher than T high, neither fragment will
form the hairpin, and both will have the same mobility. Only at temperatures within
the range of Tlow - T high will the base-substituted DNA be distinguishable from the
wild-type DNA.
73
TGGE System
By virtue of the temperature gradient which TGGE imposes upon the gel, one
particular area of the gel will provide the appropriate temperature range (i.e., Tlow Thigh) to allow formation of the hairpin, and hence, visualization of differences in
mobility.
9.2 DNA sample preparation
Add 3 µl of 95% formamide/10 mM EDTA to a 3µl aliquot of the PCR-amplified
sample. Heat to 90°C for 5 min. Immediately chill on ice!!!!!!!!!
Note: For denaturation, please refer to the above mentioned protocol. Do not use
NaOH!!!!!
9.3 Gel casting
Recipe for 10 ml gel solution (3 – 4 gels) for SSCP-ME running buffer:
Acrylamide/bis Acrylamide
Stock solution (30 : 0.8), 40% (w/v)
50x conc. SSCP-ME-buffer
(cEnd= 1 x conc.)
40% Glycerol (cEnd= 2%)
Water, distilled
3% Gel
0.75 ml
5% Gel
1.25 ml
8% Gel
2.0 ml
0.2 ml
0.2 ml
0.2 ml
0.5 ml
2 ml
0.5 ml
1.5 ml
0.5 ml
1 ml
Make sure that the urea has been completely resolved.
It is possible to heat up the urea containing solution slightly (40°C – 50°C) for a
short time in order to improve the solubilization of urea.
De-gas the solution under gentle vacuum for 3 - 5 min.
Water, distilled
TEMED
APS
17 µl
76 µl
fill up to 10 ml
17 µl
17 µl
76 µl
76 µl
Mix gently. Avoid air bubbles!
Pour the gel solution into the glass plate sandwich immediately thereafter (see
chapter 4.1.2) without air bubbles.
Cast the TGGE gels according to the instructions given under "Setting up
polyacrylamide gels".
TGGE System
74
TGGE System
75
9.4 Electrophoresis
Perpendicular TGGE with a 12 or 18 slot gel (as normally used for parallel TGGE) is
the fastest approach to determine the optimal conditions for SSCP.
Proceed as described for parallel TGGE. Establish the temperature gradient from
5°C (cathode, black) to 30°C (anode, red) and use SSCP-ME buffer for the run. Run
at 200 V for 2 - 5 hours. Covering the gel with a protective cellophane sheet is not
absolutely necessary because the gel will not dessicate at the temperatures used in
this protocol.
Silver stain the gel.
9.5 Routine analysis
When the temperature range of SSCP separation has been determined, routine
analysis can be performed in a gel with constant temperature.
TGGE System
10
76
TGGE in RNA analysis
TGGE is a perfect tool to analyze RNA for secondary structures. Applications have
been published on the differentiation of plant pathogen variants (1, 33, 34 35, 36, 37),
analysis of intermediates of plant pathogens (1, 11, 30, 31, 32, 33), and also on the
analysis of hairpin structures in m-RNA (1, 38). The technique of choice is
perpendicular TGGE. Depending on the species of RNA (M-RNA, r-RNA etc.) which
is to be analyzed, standard protocols and conditions described in this manual are
generally applicable, but may require minor modifications.
10.1 Completely double-stranded RNA
The Tm values of a dsRNA sequence will be about 20°C higher in comparison to the
corresponding ds-DNA sequence. For dsRNA with low GC-content, start with the
standard protocols using ME-buffer. For very GC-rich sequences, raise the
temperature in the TGGE gel (40°C - 80°C) or lower the ionic strength in the
electrophoresis buffer and gel by using another buffering system (electrophoresis
buffer: 0.1 x conc. TBE: 8.9 mM Tris, 8.9 mM boric acid. 0.24 mM EDTA; gel: 0.1 x
conc. TBE buffer, 5% polyacrylamide, 8 M urea; temperature gradient 35 - 60°C;
published for analysis of dsCARNA5 (1, 33, 34, 36, 37) and reovirus RNA (34)).
Note: The reduction in ionic strength will lower the Tm!!!
10.2 Partly double-stranded RNA, e.g. viroid RNA
Use the suggested protocols provided in this manual for ME-buffer or refer to the
buffer and gel systems published in various papers (electrophoresis buffer: 0.2 x
conc. TBE: 17.8 mM Tris, 17.8 mM boric acid, 0.4 mM EDTA; gel: 0.2 x conc. TBE,
5% polyacrylamide, no urea (1, 32, 33, 34, 37)).
10.3 Single-stranded RNA with single hairpin structures, m-RNA
secondary structures
Use standard protocols given in this manual for ME-buffer or refer to other buffer and
gel systems described in the literature(38) (electrophoresis buffer: 10 mM sodium
phosphate,
pH = 6.0, with or without 1 mM MgCl2; gel: 8% polyacrylamide, 10 mM sodium
phosphate, pH = 6.0, with or without 1 mM MgCl2).
TGGE System
77
10.4 Staining
For detection of the RNA, silver-staining is recommended. For identification of
double-stranded virus RNA from crude plant extracts, a protocol based on
immunoblotting has been published (35).
TGGE System
11
78
TGGE in protein analysis
TGGE can be successful applied to investigation of protein/Structural transitions, and
also thermostability of protein-nucleic acid interactions. In comparison to
conventional methods such as spectroscopy, hydrodynamics or calometry, TGGE
analysis offers several advantages:
• Only minimal amounts of sample material are required.
• TGGE may be carried out by using crude protein extracts.
• The effect of additives that influence the protein stability may easily be
investigated.
11.1 Buffers
In contrast to nucleic acids, which can generally all be analyzed with standard buffer
conditions, each protein requires its own special buffer system. This buffer has to
fulfill the same requirements as those for native gel electrophoresis of proteins:
• The protein has to be in its native conformation at low temperature (e.g. room
temperature).
• The protein has to carry a net charge.
• The protein has to be soluble in the used buffer.
• The protein has to migrate as a honogeneous band under native conditions.
This single one prerequisite has to be tested on a non denaturing gel before testing
the denaturation behavior on TGGE.
Additionally:
• The pH value of buffer should be virtually independent of the temperature.
• In order to avoid excessive current in TGGE gel above 100 mA, the ionic strength
of the electrophoresis buffer should not exceed 30 mM.
79
TGGE System
Temperature dependence of the pH-value of different electrophoresis buffers:
Buffer
pH (20°C)*
∆pH / ∆T 50°C**
----------------------------------------------------------------------------------------------------------------15 mM glycine / NaOH
30 mM H3BO3 / NaOH
40 mM Borax + 20 mM H3BO3
25 mM glycine / NaOH
30 mM Borax + 75 mM H3BO3
89 mM Tris / H3BO3
25 mM Tris / glycine
375 mM Tris / HCl
61 mM NaH2PO4 / 10 mM Na2HPO4
25 mM Na2HPO4 / 25 mM KH2PO4
30 mM Na2HPO4 / 8.7 mM KH2PO4
125 mM Tris / HCl
23 mM Na2HPO4 / 132 mM NaH2PO4
690 mM glycine + 240 mM H3BO3
48 mM Tris / H3PO4
48 mM KOH / acetic acid
20 mM sodium acetate / acetic acid
48 mM KOH / acetic acid
690 mM glycine / acetic acid
11.9
10.0
9.1
9.4
8.6
8.3
8.3
8.2
7.5
7.3
6.8
6.8
6.0
5.6
5.5
4.8
4.5
3.6
3.5
- 1.75
- 0.52
- 0.44
- 1.66
- 0.33
- 0.68
- 1.05
- 1.29
+ 0.08
- 0.07
- 0.04
- 1.39
+ 0.10
- 0.59
- 0.18
+ 0.07
+ 0.03
+ 0.09
- 0.17
___________________________________________________________________
* The pH-value of 20°C is that with the optimum buffer capacity.
** ∆pH is given for an incease in temperature of 50°C (∆T = 50°C).
In the literature, different buffer systems have been reported for the following
proteins:
Dehydrogenases (41), ß-lactamase (1), tet-repressor from E. coli (33, 42), alphaamylases (1, 34, 39), and serine proteases (40).
80
TGGE System
12
Trouble-shooting
The following trouble-shooting guide may be helpful in solving any problem that you
may encounter. If you need further assistance, please do not hesitate to contact your
local Biometra distributor or Biometra directly.
In any case where you recognize a failure which is marked in the list by an
exclamation mark, please stop working with the instrument and call the local
representative for replacing faulty parts.
Problem
Preparing
the gel solution
Urea can not be
dissolved in gel solution
Gel does not polymerize
Cause
1. Dissolving urea is an
endothermic process
and requires energy in
form of heat.
1. Heat up the acrylamide/urea
solution – but not more than 40°C
– 50°C. Mix the solution.
1. Old chemicals.
1. Prepare acrylamide/bisacrylamide solution freshly.
Prepare 4% APS freshly and
freeze in small aliquots.
2. Check all reagents that have been
included in the gel solution and
mix thoroughly.
3. Degas solution before adding
TEMED and APS.
2. Gel solution prepared
incorrectly.
3. To much oxygen in the
gel solution.
Gel polymerizes to fast
Solutions
1. To much TEMED and
APS has been added to
the gel solution.
2. The gel solution has
been heated in order to
dissolve the urea.
1. Check the concentrations of
TEMED and APS. Use the
amounts given in the standard
protocol.
2. Allow the gel solution to cool
down to room temperature before
adding TEMED and APS. (Note:
the gel solution should not be
warmed up to more than 50°C.)
81
TGGE System
Preparing
the gel sandwich
Gel solution leaks out of
sandwich
1. Gel sandwich has been
set up incorrectly
2. Clips not correct
positioned.
3. Scratches on the
spacers or old clips.
1. Clean spacers with methanol.
2. Fasten the clips above the spacer
to increase the pressure.
Polybond film with gel can not
easily removed from the
sandwich
1. Gel sticks to the glass
plate with spacer
1. Glass plate with spacer must be
treated with Acryl-Glide before
use.
Gel does not stick to the
Polybond film
1. Gel has been poured
onto the hydrophobic
side of the Polybond
film.
1. Pour the gel onto the hydrophilic
side if you want to link the gel
covalently to the Polybond film.
Hydrophobic face of the Polybond
film must face the bonding plate.
(Check the Polybond film with a
drop of water for the hydrophilic
and hydrophobic side.)
Front (top) of gel shows a zig
zag line
1. Gel has not been
overlaid with solution.
1. Overlay the gel solution in the
sandwich with 200µl Isopropyl- or
Isobutyl-Alcohol. (Alternatively bidistilled water can be used.)
Air bubbles in the gel
1. The glass plate has not
been cleaned carefully.
Air bubbles between slots
1. The glass plate needs
treatment with AcrylGlide
1. Treat glass plate with spacer with
Acryl-Glide before each use.
(Clean spacers with EtOH!)
Slots are distorted
1. Gel sticks to the slot
former and/or glass
plate.
2. Polybond film with gel
have been removed
beginning from the
bottom or to quick.
1. Treat glass plate with spacer with
Acryl-Glide before each use.
3. Use silicone grease along the
glass spacer, but never on the
sample slots!!!!
1. Clean the glass plate and slot
formers with ddH2O and EtOH
before use. Avoid the intensive
use of organic solvents. They will
dissolve the glue of the spacer
and slot formers and thus remove
them from the glass plate.
2. Sandwich was hold
2. Hold the sandwich at an angle of
vertical during pouring.
45° during pouring.
3. Solution poured to quick 3. Pour the gel solution slowly along
or in the middle of the
one side of the glass plate.
sandwich.
4. The glass plate needs
4. Treat glass plate with spacer with
treatment with AcrylAcryl-Glide
Glide
2. Remove slowly the Polybond film
with gel from the glass plate with
spacer beginning from the top.
82
TGGE System
Problem
Cause
Solutions
Electrophoresis unit
and Controller
Scratches in the white cover
film of the gradient block
1. Cover film of the
1. Remove the cover film and
gradient block damaged.
replace by a new one.
Gel running
No or minimal current
(< 5 mA), Marker dyes stop in
the gel.
1. Safety lid is not seated
properly.
2. Assembly of the TGGE
system is incorrect.
3. Gel is drying out. White
opaque areas can be
seen inside the gel.
4. Electrodes are dirty or
damaged.
5. Wicks to dry and/or
placed not correct.
6. Programmed voltage to
low.
Extremely high current
(> 50mA)
1. High ionic strength in
electrophoresis buffer,
wrong buffer
concentration.
1. Position the safety lid correctly.
2. Check the assembly of the TGGE
system and check plug
connections.
3. Carefully protect the gel against
evaporation. Take special care of
the slots. After the sample has
migrated into the gel, cover the
gel with cover film and additionally
with the special Cover glass plate
(with 2 silicone barriers).
4. Check/clean the electrodes inside
the buffer chambers.
5. Immerse the electrophoresis
wicks in the buffer and place them
properly on the gel.
6. Increase voltage.
1. Check the composition of
electrophoresis buffer and gel.
Silver staining artifacts
Cloudy yellow or brown
staining
Brown spots in the gel
1. Wash extensively after silver
binding step.
2. Use rocking table for staining
protocols.
3. Place gel up side in the staining
tray – use sufficient solution.
1. Un-dissolved crystals of 1. Dissolve urea completely before
urea remain in the gel
gel pouring.
2. Contaminated chemicals 2. Use fresh stock solutions. Filter
prior to use. Wear only nonpowdered gloves during handling
the gel.
3. Gel casting glass plate
3. Clean gel casting plate and the
or Cover film have not
Cover film carefully before they
been cleaned properly.
come in direct contact with the
gel. Wear gloves when handling
the gel.
83
TGGE System
Problem
Cause
Solutions
Gel is stained completely black 1. Chloride ions are
1. a) Check if tap water has been
or looks like a "silver mirror"
contaminating the
used for one of the buffers (tap
staining solutions, the
water always contains chloride
electrophoresis buffer or
ions). Prepare and use only fresh
the gel solution. During
solutions with ddH2O. b) Check, if
tap water has been used for
the staining protocol
washing the gel twice after
silver chloride (AgCl) is
incubation with 0.1% AgNO3 in
precipitated in the gel
dest. H2O. Use ddH2O instead. c)
(gel looks "milky" and is
If deionized water is used, its
then reduced to
elemental silver
integrity should be checked (no
chlorid ions): Take approximately
1 ml of the deionized water or the
buffer you want to check and add
some drops of 0.1% AgNO3. If
you see a milky precipitation
(silver chloride, AgCl), the solution
is contaminated with chloride
ions. Use distilled water for
preparing the buffers and washing
gel.
2. High amounts of
2. Desalt the sample prior to TGGE.
chloride ions
contaminate the sample.
Gel has a heavy background
No DNA bands are visible in
the gel
Gel fades out
1. Heavy background,
caused by smearing of
the sample: High
amounts of proteins or
polysaccharides (also
stained by the silver)
may contaminate the
sample.
2. Old chemicals,
especially
acrylamide/bis solution
3. Silver-staining protocol
has been carried out
incorrectly.
1. Check the purity of the sample
prior to TGGE.
1. No DNA, or amount of
DNA sample is below
the level of detection.
2. Too much DNA, inverse
silver-staining.
1. Check the amounts of DNA.
1. Stopping solution
(0.75% Na2CO3) was
not sufficient.
1. Incubate the gel in the staining
buffer for 10 min.
2. Use fresh stock solutions.
3. Follow the silver staining protocol
as exactly as described. Use an
excess of freshly distilled water
when washing the gel twice. Do
not prolong the incubation in
Developing solution.
2. If bands contain high amounts of
DNA, the silver-staining may
result in an inverse staining: the
background is darker than the
band itself. Reduce amounts of
DNA.
84
TGGE System
Problem
Cause
Solutions
Band pattern in general
Bands are diffuse
1. Reduce amount of DNA/RNA
1. Sample volume is too
large.
2. Sample is contaminated. 2. Purify the sample in order to
reduce contaminating proteins or
polysaccharides
3. The DNA has
3. Proceed with silver staining
undergone diffusion
protocol immediately after
inside the gel, because
electrophoresis.
the DNA has not been
fixed after running the
gel.
Interpretation of the
TGGE band pattern
DNA bands are diffuse
1. Sample volume is too
large.
2. Too much DNA in the
sample, gel is
overloaded.
3. Temperature gradient
has not been stable
during electrophoresis.
4. The gel has been shifted
during electrophoresis,
thus the temperature
gradient inside the gel
has not been stable.
5. The DNA has
undergone diffusion
inside the gel, because
of extremely prolonged
electrophoresis time at
low voltage.
6. The DNA has
undergone diffusion
inside the gel, because
the DNA has not been
fixed after running the
gel.
1. Load the correct sample volumes.
Only in parallel TGGE:
7. The band has migrated
to a temperature which
causes an irreversible
transition of the DNA
into single strands.
7. Check the melting behavior of
your DNA fragment in a
perpendicular TGGE gel. If
perpendicular TGGE shows a
sigmoidal (S-shaped) curve,
determine the effective range of
separation. Set up new conditions
for parallel TGGE. If parallel
TGGE does not show a sigmoidal
(S-shaped) curve, check all items
listed under "No S-shaped curve
in perpendicular TGGE".
2. Check the amount of DNA.
3. Check the thermal coupling
solution used. Use 0.1% Triton or
Tween 20.
4. Check the volume of thermal
coupling solution used. The gel
should not change the position
during the run.
5. Run the electrophoresis at 200 300 V (depending on the buffer
used).
6. Incubate the gel directly after the
run in Fixation solution (of the
silver staining protocol, 10%
EtOH/0.5% acetic acid). Silver
stain.
85
TGGE System
Problem
Band pattern is disturbed or
distorted
Cause
1. Air bubbles in the gel.
2.
3.
4.
5.
No S-shaped curve in
perpendicular TGGE (fig. 25)
Solutions
1. Run a new gel without air
bubbles.
Gel has been punctured 2. Load the sample carefully. Don't
(pipette tip) during
touch the gel by the pipette tip.
loading of the sample.
The edges of the gel
3. Carefully protect the gel against
have dried out during
evaporation. Cover the gel
electrophoresis. The
additionally with the special Cover
front with the bands
glass plate (with 2 silicone
"smiles".
barriers).
Wrong composition of
4. Check the composition of the gel
gel and / or
and electrophoresis buffer.
electrophoresis buffer. A
"salt front" is in the gel.
During electrophoresis
run this salt front is
indicated by an
abnormal mobility of the
marker dyes. The dye
bands look extremely
sharp, sometimes the
bromophenolblue band
moves at the same
position as the xylene
cyanol blue band.
High amounts of salt
5. Desalt the sample before loading.
ions in the sample.
Symptoms like
described under 4.
1. Wrong buffer
concentration, ionic
strength in the gel is too
high. DNA has been
denatured.
2. Amount of urea in the
gel is not sufficient. The
DNA has not been
denatured.
1. Check the composition of the gel
and the electrophoresis buffer.
2. Check the amount of urea added
to the gel. The standard protocol
requires 8 M urea when dsDNA
fragment (GC-contnent 55 - 75%)
is analyzed on TGGE.
3. Check the denaturation /
3. Ineffective renaturation
renaturation protocol used to form
of the DNA, only ssDNA
heteroduplices.
has been loaded onto
the gel.
4. Consider adding a stabilizing
4. DNA fragment is not
clamp to the fragment. Evaluate
stabilized by a GC-rich
the optimized DNA fragment for
part of the sequence.
TGGE analysis by calculating the
Irreversible melting in a
melting pattern of the sequence
one-step transition into
with the POLAND program.
completely singlestranded DNA.
5. Check the gradient block.
5. Unstable temperature
Purge any air bubbles from the
gradient or no
gradient block.
temperature gradient at
all.
TGGE System
Figure 25: No S-shaped curve in perpendicular TGGE
86
87
TGGE System
Problem
PCR product derived from a
putative heterozygous locus
exhibits no heteroduplex bands
in parallel TGGE (fig. 26 a and
b).
Cause
Solutions
1.
Electrophoresis time is
too short.
1.
2.
The temperature gradient
range used in the
experiment is not
sufficient. The
temperature at the
cathode (-) is too high.
The melting domain
containing the point
mutation has already
been denatured, when
the DNA enters the gel.
Masking of homoduplex
bands. Only heteroduplex
bands are visible in the
gel. The DNA fragments
have already passed the
"effective range of
separation". Homo- and
heteroduplices have been
separated, but the
fastetst running
homoduplex bands have
also passed "Tdiss", the
temperature of total
denaturation. Due to the
irreversible transition into
completely ssDNA, the
homoduplex bands
become diffuse,
sometimes nearly
invisible.
The sample loaded onto
the gel only contains two
different kinds of only
homoduplices with nearly
identical Tm.
The point mutation is
located in one of the most
stable melting domains
(parts) of the fragment.
2.
Run a perpendicular TGGE at first.
Estimate the effective temperature
range of separation and the running
time required for the DNA fragment
to reach this range in a parallel
TGGE run.
See 1
3.
See 1
4.
Force heteroduplex formation by
heating and reannealing the PCR
sample prior to electrophoresis.
5.
Calculate the melting map of your
DNA fragment by the POLAND
program. Construct a new fragment,
which contains the site of mutation
in one of the melting domains with
the lowest Tm values. (See
"Theoretical backbone of a
detection rate approximately
100%".)
Check for the possibility that only
one species of DNA was in your
sample: ineffective or nonexistent
PCR amplification of a particular
allele. Loss of heterozygosity in a
cell line, etc....
Check all items listed under "No Sshaped curve in perpendicular
TGGE".
3.
4.
5.
6.
6.
Only one species of DNA
has been loaded onto the
gel.
7.
Wrong composition of
buffer and/or gel,
unstable or nonexistent
temperature gradient. No
sigmoidal (S-shaped)
curve at all in the
7.
TGGE System
corresponding
perpendicular TGGE.
88
TGGE System
Figure 26 a: No heteroduplex bands in parallel TGGE
89
TGGE System
Figure 26 b: No heteroduplex bands in parallel TGGE.
90
91
TGGE System
Problem
Gel visualizes more
bands than expected (fig
27).
Cause
Solutions
1. PCR artifacts:
1. Recheck the sample by running a
Nonspecific high
non-denaturing polyacrylamide
molecular weight
gel or a perpendicular TGGE gel.
products are sometimes
Silver stain this gel. Hot start
obtained by PCR
PCR. Adjust cycle parameters to
amplification of genomic
finesse primer annealing.
DNA samples. Due to
the extremely sensitive
silver-staining they
become visible on a
TGGE gel, although the
PCR product seemed to
be "clean" on an
agarose gel stained by
ethidium bromide.
2. Check on a perpendicular gel for
2. ssDNA due to
ssDNA which does not show the
asymmetric PCR or
sigmoidal inflection. Change the
ineffective renaturation
PCR conditions in order to
during the formation of
achieve even amplification of both
heteroduplices is
single strands and force
visualized on the gel by
heteroduplex formation by heating
a band of orange to
and reannealing the PCR sample
brown-red color.
prior to electrophoresis.
3. Check the number of sigmoidal
(S-shaped) curves on a
3. DNA simply contains
perpendicular TGGE.
more species than
expected.
4. Always use glass plates slot
formers that are not damaged.
4. The slot formers on the
glass plates are
damaged. "Ghost
bands" are caused by
these imperfect slot
formers.
TGGE System
Figure 27: More bands than expected.
92
93
TGGE System
13
TGGE Testkit
13.1 Introduction
The TGGE Testkit contains samples for 4 parallel and 4 perpendicular TGGE runs
with Na-TAE buffer. The samples contain a wild type mixture with one DNA double
strand, a mutant mixture with one DNA double strand and a heteroduplex mixture
with four different DNA double strands: two homoduplices and two heteroduplices.
Wild type:
Mutant:
Heteroduplex:
Loading buffer:
min. 20 µl
min. 20 µl
min. 120 µl
min. 180 µl (with blue marker for Na-TAE buffer)
13.2 Protocol
13.2.1
Gel composition:
8% PAA
8 M Urea
0.2 x Na-TAE
2% Glycerol
Recipe for 10 ml gel solution (3 – 4 gels) for Na-TAE running buffer:
Urea (cEnd= 8 M)
Acrylamide/bis Acrylamide
stock solution (30 : 0,8), 40% (w/v)
10x conc. Na-TAE, pH 8.4 (cEnd= 0.2 x conc.)
40% Glycerol (cEnd= 2%)
Water, distilled
8% Gel
4.8 g
2.0 ml
0.2 ml
0.5 ml
2.5 ml
Make sure that the urea has been completely resolved.
It is possible to heat up the urea containing solution slightly (40°C – 50°C) for a
short time in order to improve the solubilization of urea.
De-gas the solution under gentle vacuum for 3 - 5 min.
Water, distilled
TEMED
APS (4%)
fill up to 10 ml
14 µl
45 µl
Mix gently. Avoid air bubbles!
Pour the gel solution into the glass plate sandwich immediately thereafter without
air bubbles.
TGGE System
94
13.2.2 Running buffer:
0.2 x Na-TAE
Buffer composition:
Na-TAE, pH 8.4,
1 M Sodium acetate
Stock solution (10 x conc.) 10 mM EDTA
400 mM TRIS
pH = 8.4 (titrate with Acetic Acid, never use HCl!)
Na-TAE Running Buffer
0.2x conc. Na-TAE, pH 8.4
TBE buffer: (not recommended for the TGGE Testkit)
Using TBE as running buffer results in less sharp bands, longer running time and
lower melting temperatures!
Perpendicular gel:
pre-run: 4 min + run: 40 min.
Using the 30°C - 70°C temperature gradient allows no optimization of parameters for
parallel TGGE as the melting curve starts just before the DNA is completely molten
into single strands.
Parallel gel:
pre-run: 4 min + run: 10 min.
Using the 40°C - 60°C temperature gradient gives no resolution, as all double strands
are molten and only single strands exist. The temperature gradient has to be reduced
to 20°C - 50°C. 20 min running time necessary.
95
TGGE System
13.2.3 Electrophoresis parameters:
perpendicular gel (figure 28):
Sample volume:
Temperature range (T1-T2):
Pre-run:
Run:
20µl Heteroduplex (Hd)
+ 5µl load. buffer + 25µl run. buffer
30°C - 70°C
4 min., 250 V, 20°C
40 min., 250 V, 30°C - 70°C
parallel gel (figure 29):
Sample volume:
Temperature range (T1-T2):
Pre-run:
Run:
2 µl sample
+ 0.5 µl load. buffer + 2.5 µ run. buffer
40°C - 60°C
4 min., 300 V, 25°C
10 min., 300 V, 40°C - 60°C
13.3 Gel images
This is what you should see on your gels if you proceed according to the protocol:
M
Hd
Figure 28: perpendicular gel
M
Wt
Mt
Hd
= Marker
= Wild type
= Mutant
= Heteroduplex
M
M
Mt Wt
Hd Mt
Figure 29: parallel gel
Wt Hd M
TGGE System
14
Appendix
14.1 Technical Data
14.1.1 System
Working temperature
Humidity
4°C – 35°C
10 % - 80%
14.1.2 Electrophoresis Chamber
Temperature range of gradient block
Maximum linear temperature range
(above 20°C, room temperature)
Start of linear gradient (parallel)
Start of linear gradient (perpendicular)
Temperature Accuracy
Temperature Uniformity
5°C – 80°C
45 K, for example 30°C – 75°C
or 25°C – 70°C
first marked line
end point of each line
± 0.3°C
± 0.3°C/ 2 mm
Gradient block size
Gel size (W x L)
Run distance
Size (LxWxH)
Weight
6 cm x 6 cm
7.4 cm x 8.0 cm
6.2 cm (parallel), 6.2 cm (perpendicular)
22.5 cm x 22.5 cm x 23 cm
4.2 kg
14.1.3 TGGE System Controller with integrated power pack
Program stores
100
Display
LCD
Languages
German, English
Mains voltage
115V / 230 V
Frequency
50-60 Hz
Wattage
max. 30 VA
Fuses
2x 3.15 AT (115 V) / 2x 1.6 AT (230 V)
Interfaces
1 parallel port (Centronics for printer)
1 serial port (RS232)
Size (LxWxH)
Weight
31 cm x 22 cm x 11.5 cm
3.8 kg
Integrated power pack
Voltage
Current
Power
max. 400 V
max. 500 mA
max. 30 W
96
TGGE System
14.2 Buffers
Loading buffers:
Loading buffer TBE:
0.1x conc. TBE (up to 1x conc. TBE is possible)
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
Loading buffer Na-TAE:
0.2x conc. Na-TAE, pH 8.4
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
Loading buffer ME:
10 x conc. MOPS
10 mM EDTA
0.05% Bromophenol Blue dye
0.05% Yxlene Cyanol dye
pH = 8.0
Denaturation / Renaturation (DR) Loading buffers:
DR Loading buffer TBE:
0.1x conc. TBE (up to 1x conc. TBE is possible)
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
7 M Urea
DR Loading buffer Na-TAE: 0.2x conc. Na-TAE, pH 8.4
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
8 M Urea
DR Loading buffer ME:
20 x conc. MOPS
20 mM EDTA
0.01% Bromophenol Blue dye
0.01% Yxlene Cyanol dye
8 M Urea
pH = 8.0
97
TGGE System
Running buffers:
TBE
890 mM Boric Acid
Stock solution (10 x conc.) 20 mM EDTA
890 mM TRIS
Do not titrate to adjust pH!
TBE Running buffer
0.1 x conc. TBE (up to 1x conc. TBE is possible)
Na-TAE, pH 8.4,
1 M Sodium acetate
Stock solution (10 x conc.) 10 mM EDTA
400 mM TRIS
pH = 8.4 (titrate with Acetic Acid, never use HCl!)
Na-TAE Running Buffer
0.2x conc. Na-TAE, pH 8.4
ME (MOPS/EDTA)
1 M MOPS
Stock solution (50 x conc.) 50 mM EDTA
pH = 8.0
ME-Running Buffer
1 x conc. ME, pH = 8.0
SSCP buffer:
SSCP-ME buffer
1 M MOPS
Stock solution (50 x conc) 250 mM EDTA (Free Acid)
pH = 8.0
SSCP-ME Running buffer 1 x conc. SSCP-ME, pH = 8.0
Others:
TE buffer
10 mM Tris/HCl
0.1 mM EDTA
pH = 8.0
TEMED
Solution of N,N,N’,N’tetramethylethylendiamine
APS
4% Ammonium persulfate
Glycerol 40%
Glycerol 50%
40% glycerol in water
50% glycerol in water
98
TGGE System
99
14.3 Silver staining solutions:
Standard method:
Fixation
10%
EtOH
0.5%
Acetic Acid
100 ml ethanol and 5 ml acetic acid are adjusted with
distilled water to 1 liter. Prepare freshly !
Silver Binding
0.19% AgNO3
1.9g AgNO3 is dissolved in 1 liter of distilled water. (Can
be reused for 5 gels.)
Store dark!
Developing Solution
1.5%
NaOH
0.08% NaBH4
0.1%
Formaldehyde
Dissolve 15 g NaOH in 1 liter distilled water and add 0.8g
NaBH4. Immediately before developing add 2.7 ml
formaldehyde stock solution (37% in water).
This solution must be prepared fresh every time!
Stopping Solution
0.75% Na2CO3
Dissolve 7.5 g sodium carbonate in ddH2O. Total volume:
1 liter
Quick method (for PCR products):
Fixation:
10%
EtOH
0.5%
Glacial Acid
100 ml ethanol and 5 ml acetic acid are adjusted with
double distilled water to 1 liter.
Silver Binding
0.2% AgNO3
2.0 g AgNO3 is dissolved in 1 liter of distilled water. (Can
be reused for 5 gels.)
Store dark!
Developing Solution
3.0 % NaOH
0.5%
Formaldehyde
Dissolve 3 g NaOH and 1.35 ml formaldehyde stock
solution (37% in water) in 100 ml double distilled water.
This solution must be prepared fresh every time!)
Stopping Solution:
identical with Fixation solution (10% EtOH, 0.5% Glacial
Acid)
TGGE System
100
Quick method using the AMRESCO SilverPAGE staining kit
(Code No. 211-761)
Fixation:
30%
EtOH
10%
Acetic Acid
300 ml ethanol and 100 ml acetic acid are adjusted with
double distilled water to 1 liter.
Sensibilisation:
30%
EtOH
Prepare freshly 60 ml ethanol in 140 ml double distilled
water.
Silver Binding:
Prepare Silver Binding Agent by reconstituting contents of
one pouch in 1 l of ddH20. (This solution must be
prepared fresh every time!)
Immediately before staining, add 0.7 ml of 37%
Formaldehyde to 200 ml of reconstituted Silver Binding
Agent.
Developing Solution:
Just prior to use, prepare developing solution by
reconstituting contents of one pouch of Developer I and 15
mg of Developer II in 200 ml of ddH20. (This solution
must be prepared fresh every time!)
Immediately before developing, add 0.7 ml of 37%
Formaldehyde to 200 ml of reconstituted developing
solution.
Stopping Solution:
7.5% Acetic Acid
75 ml acetic acid are adjusted with double distilled water
to 1 l.
TGGE System
15
101
References
1. Riesner, D., Henco, K. and Steger, G. (1990): Temperature-Gradient Gel
Electrophoresis: A method for the analysis of conformational transitions
and mutations in nucleic acids and protein. Page 169-250 In Chrambach,
A., Dunn, M.J., Radola, B.J.: Advances in Electrophoresis, Vol. 4, VCH
Verlagsgesellschaft Weinheim
2. Kappes, S. et al.(1995): p53 mutations in ovarian tumors, detected by
temperature-gradient gel electrophoresis, direct sequencing and
immunohistochemistry. Int. J. Cancer 64: 52-59
3. Milde-Langosch, K. et al. (1995): Presence and persistence of HPV and p53
mutation in cancer of the cervix uteri and the vulva. Int. J. Cancer 63: 639645
4. Horn, D. et al.(1996): Three novel mutations of the NF1 gene detected by
temperature gradient gel electrophoresis of exons 5 and 8.
Electrophoresis 17: 1559-1563
5. Wieland, U. et al.(1996): Quantification of HIV-1 proviral DNA and analysis of
genomic diversity b ypolymerase chain reaction and temperature gradient
gel electrophoresis. J. Virology Methods 57: 127-139
6. Kuhn, J.E. et al. (1995): Quantitation of human cytomegalovirus genomes in the
brain of AIDS patients. Journal of Medical Virology 47: 70-82
7. Linke, B. et. al. (1995): Identification and structural analysis of rearranged
immunoglobulin heavy chain genes in lymphomas and leukemia.
Leukemia 9: 840-847.
8. Menke M.A. et al. (1995): Temperature gradient gel electrophoresis for analysis of
a polymerase chain reaction-based diagnostic clonality assay in the early
stages of cutaneous T-cell lymphomas.
9. Hecker, R. et al. (1988): Analysis of RNA structure by temperature-gradient gel
electrophoresis: viroid replication and processing. Gene 72: 59-74
10. Baumstark, T. and Riesner, D. (1995): Only one of four possible secondary
structures of the central conserved region of potato spindle tuber viroid is
a substrate for processing in a potato nuclear extract. Nucleid Acids
Research 23: 4246-4254
11. Loss, P., Schmitz, M., Steger, G. and Riesner, D. (1991): Formation of a
thermodynamically metastable structure containing hairpin II is critical for
the potato spindle tuber viroid. EMBO Journal 10: 719-728
12. Riesner, D. (1998): Nucleic acid structures. In: Antisense Technology. Practical
Approach Series. Oxford University Press. p1-24 (in press)
TGGE System
102
13. Wiese, U. et al. (1995): Scanning for mutations in the human prion protein open
reading frame by temporal temperature gradient gel electrophoresis.
Electrophoresis 16: 1851-1860
14. Nubel, U. et al. (1996): Sequence heterogenities of genes encoding 16S rRNAs
in Paenibacillus polymyxa detected by temperature gradient gel
electrophoresis.
15. Lessa, E.P. and Applebaum, G. (1993): Screening techniques for detecting allelic
variation in DNA sequences. Molecular Ecology 2: 119-129
16. Richter, A., Plobner, L., Schumacher, J. 1997: Quantitatives PCR-Verfahren zur
Bestimmung
der
Plasmidkopienzahl
in
rekombinanten
Expressionssystemen. BIOforum 20: 545-547
17. Henco, K. and Heibey, M. (1990): Quantitative PCR – the determination of
template copy numbers by temperature gradient gel electrophoresis.
Nucleic Acids Research 18: 6733-6734
18. Birmes, A. et al. (1990): Analysis of the conformational transition of proteins by
temperature-gradient gel electrophoresis. Electrophoresis 11: 795-801
19. Arakawa, T. et al. (1993): Analysis of the heat-induced denaturation of proteins
using temperature gradient gel electrophoresis. Analytical Biochemistry
208: 255-259
20. Chen, X. et al. (1995): High resolution SSCP by optimization of the temperature
by transverse TGGE. Nucleic Acids Research 23: 4524-4525
21. Scholz, R.B. et al. (1993): Rapid screening for Tp53 mutations by temperature
gradient gel electrophoresis: a comparison with SSCP analysis. Human
Molecular Genetics 2: 2155-2158
22. Elphinstone and Baverstock, P.R. (1997): Detecting mitochondrial genotypes by
temperature gradient gel electrophoresis and heteroduplex analysis.
BioTechniques 23: 982-986
23. Poland, D. (1974): Recursion relation generation of probability profiles for
sequence-specific macromolecules with long-range correlations.
Biopolymers 13:1859-1871
24. Lerman, L.S. and Silverstein, K. (1987): Computational simulation of DNA-melting
and its application to denaturing gradient-gel electrophoresis. Meth.
Enzymol. 155: 482-501
25. Steger, G. (1994): Thermal denaturation of double-stranded nucleic acids:
prediction of temperatures critical for gradient electrophoresis and
polymerase chain reaction.
TGGE System
103
26. Schumacher, J, Randels, J.W. and Riesner,D. (1983): A two dimensional
electrophoretic technique for detection of circular viroids and virusoids.
Anal. Biochem. 135, 288 - 295
27. Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989): Molecular cloning, Cold
Spring Habor Laboratory press
28. Steger, G. and Riesner, D. (1992): Temperaturgradienten-Gelelektrophorese:
eine Methode zur Analyse von Konformationsübergängen und Mutationen
in Nukleinsäuren und Proteinen. In Radola, B.J. (ed) Handbuch der
elektrophorese, VCH Verlagsgesellschaft, Weinheim
29. Sheffield, V.C., Cox, D.R. and Lerman, R.M. (1989): Attachment of a 40-basepair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the
polymerase chain reactiob results in improved detection of single-base
changes. Proc. Natl. Acad. Sci. USA 86, 232 - 236
30. Hecker R., Wang Z., Steger G. and Riesner D. (1988): Analysis of RNA structure
by temperature-gradient gel electrophoresis: viroid replication and
processing. Gene 72, 59-74
31. Jiang L., Chen W., Tain L.P. and Liu Y. (1991): Temperature-gradient gel
electrophoresis of apple scar skin viroid. Acta Microbiol. Sin. 30, 278-283
32. Riesner D., Hecker R. and Steger G. (1988): Structure of viroid replication
intermediates as studied by thermodynamics and temperature-gradient gel
electrophoresis. In Sarma R.H. and Sarma M.H. (eds.) Structure &
Expression, Vol. I: From Proteins to Ribosomes, Adenine press, 261-285
33. Riesner D., Steger G., Zimmat R., Owens R.A., Wagenhöfer M., Hillen W.,
Vollbach S. and Henco K. (1989): Temperature-gradient gel
electrophoresis of nuleic acids: Analysis of confor-mational transitions,
sequence variations, and protein-nucleic acid interactions. Electrophoresis
10, 377-389
34. Rosenbaum V. and Riesner D. (1987): Temperature-gradient gel electrophoresis:
thermodynamic analysis of nucleic acids and proteins in purified form and
in cellular extract. Biohys. Chem. 26, 235-246
35. Schönborn J., Oberstraß J., Breyel E., Tittgen J., Schumacher J., Lukacs N.
(1991): Monoclonall antibodies to double-stranded RNA as probes of RNA
structure in crude nucleic acid extracts. Nucleic Acids Res. 19, 2993-3000
36. Po Tien, Steger G., Rosenbaum V., Kaper J. and Riesner D. (1987): Doublestranded cucumovirus associated RNA5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel
electrophoresis. Nucleic Acids Res. 15, 5069-5083
TGGE System
104
37. Zimmat R., Gruner R., Hecker R., Steger G. and Riesner D. (1991): Analysis of
mutations in viroid RNA by non-denaturing and temperature-gradient gel
electrophoresis. In R.H. Sarma and M.H. Sarma (eds.) Structure &
Methods, Vol. 3:, DNA & RNA, Adenine Press, 339-357
38. Rosenbaum V., Klahn T., Lundberg Holmgren E., von Gabain A. and Riesner D.
(1992): Co-existing structures of an mRNA stability determinat: The 5‘
region of the Escherichia coli and Serratia marcescens ompA mRNA,
J.Mol.Biol., in press
39. Birmes A., Sättler A., Maurer S.O. and Riesner D. (1990): „Analysis of the
conformational transitions of proteins by temperature-gradient gel
electrophoresis“. Electrophoresis 11, 795-801
40. Sättler A., Kanka S., Schrörs W. and Riesner D. (1992): „Random mutagenesis of
the weak calcium binding side in SubtilisinCarlsberg and screening for
thermal stability by temperature-gradient gel electrophoresis“. Accepted
for: 1st International Symposium of Subtilisin Enzymes, EMBL, Hamburg
41. Thatcher D. and Hodson B. (1981): „Denaturation of proteins and nucleic acids
by thermal-gradient electrophoresis“. Biochem. J. 197, 105-109
42. Wagenhöfer M., Hansen D. and Hillen W. (1988): „Thermal denaturation of
engineered tet repressor proteins and their complexes with tet operator
and tetracycline studie by temperature-gradient gel electrophoresis“.
Analytical Biochem. 175, 422-432
43. Sanguinetti C.J., Neto E.D. and Simpson A.J.G. (1994): BioTechniques 17, 915
44. Kappes, S., Milde-Langosch, K., Kressin, P., Passlack, B., Dockhorn-Dwornczak,
B., Röhlke, P. and Löning, T. (1995): "p53 Mutations in ovarian tumors,
detected by temperature-gradient gel electrophoresis, direct sequencing
and immunohistochemistry". Int. J. Cancer 64, 52 - 59
45. Kluwe, L., MacCollin, M., Tatagiba, M., Thomas, S., Hazim, W., Haase, W. and
Mautner, V.-F. (1998): "Phenotypic variability associated with 14 splicesite mutations in the NF2 gene". American Journal of Medical Genetics 77,
228 - 233
46. Lerman, L. S. and Beldjord, C. (1998). "Comprehensive mutation detection with
denaturing gradient gel electrophoresis". In R.G.H. Cotton, E. Edkins and
S. Forrest (eds.) Mutation Detection, A Practical Approach, Oxford
University Press, 35 - 62
47. Gamper, H., Piette, J. and Hearst, J.E. (1984): Photochem. Photobiol. 40, 29 ff
105
TGGE System
16
Order information and spare parts
Biometra offers a wide range of accessories parts and consumables related to
Temperature Gradient Gel Electrophoresis.
Item
Order No.
TGGE System; 230 V, electrophoresis unit with Peltier element-powered gradient
block and 2 removable electrophoresis buffer chambers, system controller with
integrated power supply, Starter Kit and manual
TGGE System, 115 V, dito
TGGE System Controller with integrated power supply, 230 V
TGGE System Controller with integrated power supply, 115 V
TGGE electrophoresis unit with Peltier element powered gradient block, 2 removable
electrophoresis buffer chambers and connector cable
TGGE connector cable (controller to electrophoresis unit)
TGGE Starter Kit with 3 Bonding glass plates, 3 types of glass plates with slots, precut electrode wicks, pre-cut Polybond film and cover film
024-000
TGGE Testkit, samples for 4 parallel and 4 perpendicular TGGE runs (20 µl wild
type, 20 µl mutant, 120 µl Heteroduplex, 180 µl loading buffer with blue-marker for
Na-TAE buffer)
Accessories
TGGE removable electrophoresis buffer chambers,
TGGE electrode wicks, pre-cut 8 x 7 cm,
2 pcs
100 pcs
TGGE bonding plate, 9 x 9 cm, w/o spacer
TGGE glas plate 9 x 9 cm, 8 slots 4x3x0.4 mm,
approx. 5 µl
TGGE glas plate 9 x 9 cm, 1 slot (rectangular) 40x3x0.4 mm, approx. 50 µl
TGGE glas plate 9 x 9 cm, 1 slot (diagonal) 62x3x0.4 mm,
approx. 75 µl
TGGE glas plate 9 x 9 cm, 12 slots 3x2x0.4 mm,
TGGE glas plate 9 x 9 cm, 18 slots 2x2x0.4 mm,
TGGE glas plate 9 x 9 cm, 0.5 mm spacer, no slots
approx. 3 µl
approx. 2 µl
TGGE Polybond film, pre-cut 8.8 x 8.8 cm,
25/pkg
TGGE Cover glass plate with silicone barriers
+ 10 cover films, pre-cut 7 x 6 cm
TGGE cover film, pre-cut 7 x 6 cm
25/pkg
TGGE Polybond film, pre-cut 8.8 x 8.8 cm,
100/pkg
TGGE cover film, pre-cut 7 x 6 cm
100/pkg
TGGE Slotformer („Slot forming units“), 10 x „multi“, 9 x „long“
Gel casting clips,
3/pkg
Consumables
Acrylamide/bis Acrylamide, 40% (30:0.8), 500 ml
Ammonium Persulfate, 4x 25 g
EDTA (Tetrasodium Salt, Dihydrate), 500 g
Glycerol, 1 l
Sodium Acetate, anhydrous, 500 g
TEMED, 25 ml
Acryl-Glide, 100 ml
Silver PAGE, Silver staining kit for 20 stains
TRIS, 1 kg
024-090
024-001
024-091
024-002
024-033
024-003
024-050
024-010
024-015
024-021
024-022
024-023
024-024
024-025
024-026
024-027
024-030
024-031
024-032
024-034
024-035
024-121
010-007
210-254
210-486
210-245
210-854
210-602
210-761
211-319
211-761
220-826
Consumables from Biometra are not available outside Germany. Please source from another supplier.
TGGE System
17
106
Instructions for return shipment
If you would like to send the unit back to us, please read the following
return instructions.
Should you have any problems with the TGGE System, please contact your local
Biometra dealer or our service department:
Biometra biomedizinische Analytik GmbH
Service Department
Rudolf-Wissell-Straße 30
D-37079 Göttingen
Phone:++49 – (0)5 51 / 50 68 6-0
Fax: ++49 – (0)5 51 / 50 68 6-66
• Return only defective devices. For technical problems which are not definitively
recognisable as device faults please contact the Technical Service Department at
Biometra.
• Use the original box or a similarly sturdy one.
• Label the outside of the box with “CAUTION! SENSITIVE INSTRUMENT!”
• Please enclose a precise description of the fault, which also reveals during
which procedures the fault occurred, if possible.
• Important: Clean all parts of the instrument from residues, and of biologically
dangerous, chemical and radioactive contaminants. Please include a
written confirmation ( use the “Equipment Decontamination
Declaration” following on the next page) that the device is free of
biologically dangerous and chemical or radioactive contaminants in
each shipment. If the device is contaminated, it is possible that
Biometra will be forced to refuse to accept the device.
• The sender of the repair order will be held liable for possible losses resulting
from insufficient decontamination of the device.
• Please enclose a note which contains the following:
a) Sender’s name and address,
b) Name of a contact person for further inquiries with telephone number.
107
TGGE System
18
Equipment Decontamination Certificate
To enable us to comply with german law (i.e. §28 StrlSchV, §17 GefStoffV and §19 ChemG) and to
avoid exposure to hazardous materials during handling or repair, will you please complete this form,
prior to the equipment leaving your laboratory
COMPANY / INSTITUTE _____________________________________________________________
ADDRESS ________________________________________________________________________
TEL NO ______________________________
FAX NO _____________________________
E-MAIL ___________________________________________________________________________
EQUIPMENT
If on loan / evaluation
Model
Serial No
______________
__________________
______________
__________________
______________
__________________
______________
__________________
Start Date: ________
Finish Date ________
Hazardous materials used with this equipment
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
Has the equipment been cleaned and decontaminated? YES / NO (delete)
Method of cleaning / decontamination
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
NAME _______________________________
POSITION ___________________________
(HEAD OF DIV./ DEP./ INSTITUTE / COMPANY)
SIGNED _____________________________
DATE _______________________________
PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA
DISTRIBUTOR TOGETHER WITH THE EQUIPMENT.
PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING. INSTRUMENTS WITHOUT
THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER.
TGGE System
19
108
Warranty
This Biometra instrument has been carefully built, inspected and quality controlled
before dispatch. Hereby Biometra warrants that this instrument conforms to the
specifications given in this manual. This warranty covers defects in materials or
workmanship for 12 month as described under the following conditions:
This warranty is valid for 12 month from date of shipment to the customer from
Biometra or an authorized distributor. This warranty will not be extended to a third
party without a written agreement of Biometra.
This warranty covers only the instrument and all original accessories delivered with
the instrument. This warranty is valid only if the instrument is operated as
described in the manual.
Biometra will repair or replace each part which is returned and found to be
defective.
This warranty does not apply to wear from normal use, failure to follow operating
instructions, negligence or to parts altered or abused.
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