Mentype® Argus Y-MHQS

Mentype® Argus Y-MHQS PCR Amplification Kit
Product description
The Mentype® Argus Y-MHQS PCR Amplification Kit is a multiplex application for the
nine Y-chromosomal Short Tandem Repeat (STR) loci of the Minimal Haplotype (MH)
standard. The primers of DYS19, DYS385ab, DYS389-I, DYS389-II, DYS390,
DYS391, DYS392, and DYS393 are fluorescence-labelled with 6-FAM or HEX.
The test kit was developed specifically for fast and reliable generation of male DNA
profiles from mixtures of male and female DNA up to a ratio of 1:6000 so that
separation of sperm from female cells or differential lysis is not required. The detection
limit of the Mentype® Argus Y-MHQS PCR Amplification Kit is up to 100 pg genomic
male DNA. However, it is recommended to use 0.1-1.0 ng male DNA.
As special feature, Mentype® Argus Y-MHQS contains an internal PCR control
(Quality Sensor “QS”) which provides helpful information on the efficiency of the PCR
and on the presence of PCR inhibitors.
The Mentype® Argus Y-MHQS was developed according to the recommendations of
the International Forensic Y-User Group (http://www.yhrd.org/index.html). This
consortium has evaluated a core set of a highly informative Y-STR, the so called
Minimal Haplotype which was recommended for court. Generation of DNA profiles
using Mentype® Argus Y-MHQS corresponds to the guidelines of the International
Society for Forensic Genetics (Gill et al., 2001 a and b; Gusmão et al., 2005a). All
important population-genetic data could be calculated with the GenoProof® Software.
The test kit was validated and evaluated using the GeneAmp® 9700 thermal cycler,
ABI PRISM® 310 Genetic Analyzer, and ABI PRISM® 3100/3130 Genetic Analyzer.
Table 1. Locus-specific information of Mentype® Argus Y-MHQS
STR Locus
DYS19
DYS385
DYS389-I
DYS389-II
DYS390
DYS391
DYS392
DYS393
GenBank®
accession
AC017019
AC022486
AC004617
AC004617
AC011289
AC011302
AC011745
AC006152
Repeat motif
of the reference allele
[TAGA]3 TAGG [TAGA]12
[GAAA]11
[TCTG]3[TCTA]9
[TCTG]5 [TCTA]12 [TCTG]3 [TCTA]9
[TCTG]8 [TCTA]11 [TCTG]1 [TCTA]4
[TCTA]11
[TAT]13
[AGAT]12
Reference
allele
15
11
12
29
24
11
13
12
Allele
range
9-19
6-28
9-17
24-35
12, 17-29
5-16
4-20
7-18
Table 1 shows the STR loci with their repeat motifs and alleles. The most frequent
alleles for European populations are included in the allelic ladder. Allele ranges include
all known alleles of YHRD (http://www.yhrd.org as at 12/2008) and of the current
literature.
2
Yp11.32
Yp11.31
DYS393
2.8 Mb
DYS19
9.2 Mb
Yp11.2
Yp11.1
Yq11.1
Yq11.21
DYS391
DYS389-I, -II
Yq11.221
DYS390
Yq11.222
DYS385
DYS392
21.5 Mb
Yq11.223
Yq11.23
Yq12
Fig. 1 The ideogram of the Y-chromosome describes the physical localisation of the STR loci which can be
analysed with Mentype® Argus Y-MHQS. The positions of the STR loci are shown in Mb (http://www.ncbi.nlm.
nih.gov/genome /guide/human as at 08/2008).
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Table 2. Chromosomal Mapping of Mentype® Argus Y-MHQS
Locus
DYS19
DYS385
DYS389-I
DYS389-II
DYS390
DYS391
DYS392
DYS393
Chromosomal mapping
Yp11.2
Yq11.222
Yq11.21
Yq11.21
Yq11.221
Yq11.21
Yq11.222
Yp11.31
Content
Mentype® Argus Y-MHQS PCR Amplification Kit (100 Reactions)
Nuclease-free water
Reaction mix B
Primer mix
Control DNA XY1 (2 ng/μL)
Control DNA XX28 (100 ng/μL)
DNA Size Standard 550 (ROX)
Allelic ladder
3.0 mL
500 μL
250 μL
10 μL
10 μL
50 μL
10 μL
Ordering information
Mentype® Argus Y-MHQS
Mentype® Argus Y-MHQS
Mentype® Argus Y-MHQS
25
100
400
Reactions
Reactions
Reactions
Cat. No.
Cat. No.
Cat. No.
42-09112-0025
42-09112-0100
42-09112-0400
Storage
Store all components at -20°C and avoid repeated thawing and freezing. Primer mix
and allelic ladder must be stored protected from light. The DNA samples and post-PCR
reagents (allelic ladder and DNA size standard) should be stored separately from the
PCR reagents. The expiry date is indicated on the kit cover.
Quality assurance
All contents of Biotype® test kits undergo an intensive quality assurance process at
Biotype AG. The quality of the test kits is permanently monitored in order to ensure
unrestricted usability. Please contact us if you have any questions regarding quality
assurance.
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Additionally required reagents
Additional reagents are needed in order to be able to use the Biotype®
PCR Amplification Kit. The use of the following products is strongly recommended:
Reagent
JumpStart™ Taq DNA Polymerase
hot start, 2.5 U/μL, 50 U oder 250 U
Hi-Di™ Formamide, 25 mL
Matrix Standards DS-30
for ABI PRISM® 310 Genetic Analyzer
Matrix Standards DS-30
for ABI PRISM® 3100/3130/3730
Supplier
Order number
Sigma-Aldrich
D4184
Applied Biosystems
4311320
Applied Biosystems
401546 und 402996 (NED)
Applied Biosystems
4345827
Trademarks and patents
Mentype® is a registered trademark of Biotype AG.
GenoProof® is a registered trademark of Qualitype AG.
JumpStart™ is a registered trademark of Sigma-Aldrich.
ABI PRISM®, GeneScan®, Genotyper®, GeneMapper™ and Applied Biosystems are
registered trademarks of Applied Biosystems Inc. or its subsidiaries in the U.S. and
certain other countries.
6-FAM, HEX, NED, ROX, POP-4 and Hi-Di are trademarks of Applied Biosystems Inc.
GeneAmp® is a registered trademark of Roche Molecular Systems.
The PCR is covered by patents. Patentees are Hoffmann-La Roche Inc. and
F. Hoffmann-La Roche (Roche).
GenBank® is a trademark of National Institute of Health.
Warnings and safety instructions
The PCR Amplification Kit contains the following potentially hazardous chemicals:
Kit component
Primer mix, reaction mix
and allelic ladder
Chemical
Sodium azide NaN3
Hazards
Very toxic if swallowed, develops toxic
gases when it gets in contact with acids
Observe the Material Safety Data Sheets (MSDS) for all Biotype® products, which are
available on request. Please contact the respective manufacturers for copies of the
MSDS for any additionally needed reagents.
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Content
1. PCR amplification...........................................................................................6
1.1 Master mix preparation ............................................................................6
1.2 PCR amplification parameter ....................................................................7
2. Electrophoresis using the ABI PRISM® 310 Genetic Analyzer ............................8
2.1 Matrix generation.....................................................................................8
2.2 Sample preparation................................................................................11
2.3 Setting up the GeneScan® software ........................................................11
2.4 Analysis parameter ................................................................................12
3. Electrophoresis using the ABI PRISM® 3130/3130xl Genetic Analyzer.............13
3.1 Spectral calibration / matrix generation ...................................................13
3.2 Sample preparation................................................................................16
3.3 Setting up the GeneMapper™ ID software ..............................................17
3.4 Analysis parameter / analysis method .....................................................19
4. Analysis.......................................................................................................20
4.1 Biotype® template files...........................................................................22
4.2 Controls ................................................................................................23
4.3 Lengths of fragments and alleles ............................................................23
5. Interpretation of results ................................................................................28
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Protocols for PCR amplification, electrophoresis, and analysis
1. PCR amplification
1.1 Master mix preparation
The table below shows the volumes of all PCR reagents per 25 μL reaction volume,
including a sample volume of 3.0 μL (template DNA). The number of reactions to be
set up shall be determined taking into account positive and negative control reactions.
Add one or two reactions to this number to compensate the pipetting error.
Data in [μL]
Nuclease-free water
Reaction mix B*
Primer mix
Taq DNA Polymerase (hot start, 2.5 U/μL)
Volume of master mix
1
14.1
5.0
2.5
0.4
22.0
Number of PCR samples
10
25
141.0
352.5
50.0
125.0
25.0
62.5
4.0
10.0
220.0
550.0
100
1410.0
500.0
250.0
40.0
2200.0
* contains Mg2+, dNTP Mix, BSA
All components should be mixed (vortex) and centrifuged for about 10 s before
preparing the master mix. The DNA volume applied to the assay depends on its
concentration. A volume of up to 5 μL may be necessary for DNA trace templates. DNA
volumes of more than 5 μL are not recommended, because potential PCR inhibitors
may interfere with the process. Fill up the final reaction volume to 25 μL with
nuclease-free water.
Generally, DNA templates shall be stored in nuclease-free water or in diluted TE buffer
(10 mM Tris HCl, pH 8.0 and 1 mM EDTA), e.g. 0.1x TE buffer.
The primer mixes are adjusted for balanced peak heights at 30 PCR cycles and
0.5 ng Control DNA XY1 in a reaction volume of 25 μL. If more DNA template is
introduced, higher peaks can be expected for small PCR fragments and relatively low
peaks for large fragments. Reduce the amount of DNA template to correct this
imbalance.
Positive control
For the positive amplification control, dilute the Control DNA XY1 to 0.5 ng in the
appropriate volume. Instead of the template DNA, pipette the diluted Control DNA into a
reaction tube containing the PCR master mix.
Negative control
For the negative amplification control, pipette nuclease-free water instead of template
DNA into a reaction tube which contains the PCR master mix.
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1.2 PCR amplification parameter
Perform a “hot start” PCR in order to activate the Taq DNA Polymerase and to prevent
the formation of non-specific amplification products.
The number of cycles depends on the amount of DNA. 30 cycles are recommended for
all samples. 34 cycles are recommended optionally in order to achieve optimal signal
intensities for stains with small amounts of genomic DNA.
Standard method
Recommended for all DNA samples
Temperature
94°C
94°C
58°C
72°C
68°C
10°C
Time
4 min (hot start for activation of the JumpStart™ Taq DNA Polymerase)
30 s
30 cycles
120 s
75 s
60 min
∞
hold
Optional
Recommended for stains with small amounts of genomic DNA
Temperature
94°C
94°C
58°C
72°C
68°C
10°C
Time
4 min (hot start for activation of the JumpStart™ Taq DNA Polymerase)
30 s
34 cycles
120 s
75 s
60 min
∞
hold
Small amounts of DNA may result in allelic dropouts and imbalances of the peaks.
Furthermore, unspecific amplification products could appear. With increasing numbers
of cycles, there is the risk of cross contamination caused by minimal amounts of
impurities.
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2. Electrophoresis using the ABI PRISM® 310 Genetic Analyzer
For general instructions on instrument setup, matrix generation and application of the
GeneScan® or GeneMapper™ ID software, refer to the ABI PRISM ® 310 Genetic
Analyzer User’s Manual. Electrophoresis using the GeneScan® software is described
below.
The virtual filter set D shall be used for combined application of the four fluorescent
labels 6-FAM, HEX, NED, and ROX (also called DS-30). Generally, Filter Sets A and F
are suitable, too.
Material
Capillary
Polymer
Buffer
47 cm / 50 μm (green)
POP-4 for 310 Genetic Analyzer
10x Genetic Analyzer Buffer with EDTA
2.1 Matrix generation
Prior to conducting DNA fragment size analysis with the filter set D, a matrix with the
four fluorescent labels 6-FAM, HEX, NED, and ROX must be generated. The suitable
matrix standard DS-30 is available from Applied Biosystems.
Colour
Blue (B)
Green (G)
Yellow (Y)
Red (R)
Matrix standard
6-FAM
HEX
NED
ROX
Order number
Applied Biosystems, 401546
Applied Biosystems, 401546
Applied Biosystems, 402996
Applied Biosystems, 401546
Four electrophoresis runs shall be conducted, one for each fluorescent label, 6-FAM,
HEX, NED, and ROX, under the same conditions as for the samples and allelic ladders
of the Biotype® test kit to generate suitable matrix files.
Matrix sample
Composition
Hi-Di™ Formamide
Matrix standard 6-FAM
Volume
12.0 μL
1.0 μL
Matrix sample 2
Hi-Di™ Formamide
Matrix standard HEX
12.0 μL
1.0 μL
Matrix sample 3
Hi-Di™ Formamide
Matrix standard NED
12.0 μL
1.0 μL
Matrix sample 4
Hi-Di™ Formamide
Matrix standard ROX
12.0 μL
1.0 μL
Matrix sample 1
- Denaturation for 3 min at 95°C
- Cool down to 4°C
- For analysis: load the samples on the tray
- Create a Sample Sheet and enter sample designation
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Injection list for matrix generation
Injection list
Module File
Matrix File
Size Standard*
Injection [s]
Injection [kV]
Run [kV]
Run [°C]
Run Time [min]
GS STR POP-4 (1 mL) D
NONE
NONE
5
15.0
15.0
60
24
* prepare matrix standards always without DNA Size Standard (ROX)
Analysis of the matrix samples
- Run the GeneScan® software
- File → New → Project (open folder of current run) → Add Sample Files
- Select a matrix sample in the Sample File column
- Sample → Raw Data
- Check the matrix samples regarding a flat baseline. As shown in the figure below,
there should be at least five peaks with peak heights about 400-4000 (Y-axis) for
each matrix sample (optimal range: 1000-3000)
▼ 3400 Data Points (X)
6400▼
Fig. 2 Electropherogram with raw data of the matrix standard 6-FAM
- Select analysis range with flat baseline and re-inject the matrix sample if necessary
- Note down start and end value (data points) of the analysis range, e.g.
start value 3400, end value 6400
- Calculate the difference, e.g. 6400-3400 = 3000 data points
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Generation of a new matrix
- File → New → Matrix
Fig. 3 Matrix sample selection
- Import matrix samples for all dyes (B, G, Y, R)
- Enter a Start At value, e.g. 3400
- Enter the calculated difference under Points, e.g. 3000
- Click on OK to calculate the new matrix
Fig. 4 New matrix DS-30
- Save the matrix in the matrix folder: File → Save as, e.g. Matrix DS-30
Matrix check
Check the new matrix with current samples.
- File → New → Project (open folder of the respective run) → Add Sample Files
- Select sample(s) in the Sample File column
- Sample → Install New Matrix (open matrix folder and select new matrix)
- Re-analyse your samples
There should be no pull-up peaks between the dye panels (B, G, Y, R) with the new
matrix.
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2.2 Sample preparation
Composition
Volume
Hi-Di™ Formamide
12.0 μL
DNA Size Standard 550 (ROX)
0.5 μL
prepare 12 μL of the mix (formamide + DNA size standard) for all samples
add 1 μL PCR product (diluted if necessary) or allelic ladder
- Denaturation for 3 min at 95°C
- Cool down to 4°C
- For analysis: load the samples on the tray
Signal intensities
Options to increase the signal intensity:
- Reduce the volume of the DNA Size Standard 550 (ROX) to peak heights of
about 500 relative fluorescent units (RFU)
- Purify the PCR products before starting the analysis
2.3 Setting up the GeneScan® software
- Create a Sample sheet and enter sample designation
Injection list
Module File
Matrix File
Size Standard
Injection [s]*
Injection [kV]
Run [kV]
Run [°C]
Run Time [min]**
GS STR POP-4 (1 mL) D
e.g. Matrix Biotype
e.g. SST-ROX_50-400bp
5
15.0
15.0
60
26
* Deviating from standard settings, the injection time may range between 1 and 10 s depending on the type of
sample. If blood samples with very high signal intensities are recorded, a shorter injection time may be selected.
For samples with low DNA content an injection time up to 10 s may be necessary.
** Depending on the analysis conditions the run time for Mentype® Argus Y-MHQS was modified in order to
analyse fragments with lengths of up to 400 bp.
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2.4 Analysis parameter
The recommended analysis parameters are:
Analysis Range
Data Processing
Peak Detection
Size Call Range
Size Calling Method
Split Peak Correction
Start: 2000
Stop: 10000
Baseline: Checked
Multicomponent: Checked
Smooth Options: Light
Peak Amplitude Thresholds
B:* Y:*
G:* R:*
Min. Peak Half Width: 2 pts
Polynorminal Degree: 3
Peak Window Size: 11 pts**
Min: 50
Max: 550
Local Southern Method
None
* The peak amplitude threshold (cutoff value) corresponds to the minimum peak height that will be detected by
the GeneScan® or GeneMapper™ ID software. Thresholds are usually 50-200 RFU and should be determined
individually by the laboratory. Recommendation: The minimal peak height should be three times as high as the
background noise of the baseline.
** Point alleles (i.e. alleles with at least 1 bp difference to the next integer allele) may occasionally not be
distinguished. For improved peak detection, minimise the Peak Window Size further.
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3. Electrophoresis using the ABI PRISM® 3130/3130xl Genetic Analyzer
For detailed instructions on instrument setup, spectral calibration, or application of the
ABI PRISM® Data Collection software version 3.0 and the GeneMapper™ ID software,
refer to the ABI PRISM ® 3130/3130xl Genetic Analyzers Getting Started Guide.
Electrophoresis on an ABI PRISM® 3130 Genetic Analyser by using the GeneMapper™
ID software is described below.
The system with 4 capillaries is named ABI 3130 (former ABI 3100-Avant), and the
system with 16 capillaries is named ABI 3130xl (former ABI 3100).
The virtual filter set D shall be used for combined application of the four fluorescent
labels 6-FAM, HEX, NED, and ROX (also called DS-30).
Material
Capillary
Polymer
Buffer
36 cm Capillary Array for 3130/3130xl
POP-4 Polymer for 3130
10x Genetic Analyzer Buffer with EDTA
3.1 Spectral calibration / matrix generation
Prior to conducting DNA fragment size analysis, it is necessary to perform a spectral
calibration with the four fluorescent labels 6-FAM, HEX, NED, and ROX for each
analyzer. The calibration procedure creates a matrix which is used to correct the
overlapping of fluorescence emission spectra of the dyes.
Spectral calibration comprises the following steps:
- Preparation the spectral calibration standards
- Loading the standards to the 96-well reaction plate (one sample per capillary)
- Creating the instrument protocol for spectral calibration (Protocol Manager)
- Defining the plate composition in the plate editor (Plate Manager)
- Performing a spectral calibration run and checking the matrix
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Setting up the spectral calibration standards
Example for 4 capillaries/ABI 3130
Composition
Volume
Hi-Di™ Formamide
47.5 μL
Matrix standard DS-30
2.5 μL
- Denaturation for 3 min at 95°C
- Cool down to 4°C
- For analysis, load 10 μL of the matrix standard into a 96-well reaction plate, well A1-D1
Example for 16 Capillaries/ABI 3130xl
Composition
Volume
Hi-Di™ Formamide
190 μL
Matrix standard DS-30
10.0 μL
- Denaturation for 3 min at 95°C
- Cool down to 4°C
- For analysis, load 10 μL of the matrix standard into a 96-well reaction plate, well A1-H1 and A2-H2
Performing a spectral calibration run
- Insert the 96-well plate on the autosampler tray
- In the Protocol Manager of the Data Collection Software click New the window
Instrument Protocol to open the Protocol Editor dialog box
Instrument Protocol for spectral calibration
Protocol Editor
Name
Type
Dye Set
Polymer
Array Length
Chemistry
Run Module
e.g. Spectral36_POP4_DS30
SPECTRAL
D
POP4
36
Matrix Standard
Spect36_POP4_1
- Select OK to complete the Protocol Editor dialog box
- In the Plate Manager of the Data Collection software click New to open the New
Plate Dialog box
Plate Editor for spectral calibration (I)
New Plate Dialog
Name
Application
Plate Type
Owner Name / Operator Name
e.g. Spectral_DS-30
Spectral Calibration
96-Well
…
- Select OK. A new table in the Plate Editor opens automatically
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Plate Editor for spectral calibration (II)
Column
Sample Name
Priority
Instrument Protocol 1
Type name for the matrix samples
e.g. 100
Spectral36_POP4_DS30 (setting described before)
- Click into the column header to select the entire column, select Edit → Fill Down to
apply the information to all selected samples, and click on OK
- In the Run Scheduler click on Find All, select Link to link the reaction plate on the
autosampler up with the newly created plate record (position A or B) and start the run
R, Y, G,
B
Fig. 5 Electropherogram of spectral calibration with matrix standard DS-30
Matrix check
- The quality value (Q value) of each capillary must be greater than 0.95 and the
condition number range (C value) must be between 1 and 20.
- Check the matrix samples for a flat baseline. As shown in Fig. 5, there should be four
peaks with peak heights of about 1000-5000 (Y-axis) in each matrix sample (optimal
range: 2000-4000)
- Check the new matrix with your current samples. There should be no pull-up peaks
between the dye panels (B, G, Y, R, O) with the new matrix
- If calibration was not successful, use the optimised values and repeat the calibration
run
- If all capillaries have passed the test, the last calibration file for the Dye Set D is
activated automatically in the Spectral Viewer. Rename the calibration file (e.g.
DS-30_Date of calibration) using the respective button
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3.2 Sample preparation
Composition
Volume
Hi-Di™ Formamide
12.0 μL
DNA Size Standard 550 (ROX)
0.5 μL
prepare 12 μL of the mix (formamide + DNA size standard) for all samples
add 1 μL PCR product (diluted if necessary) or allelic ladder
- Denaturation for 3 min at 95°C
- Cool down to 4°C
- For analysis: load the samples on the tray
Because injections take place simultaneously on all capillaries, four samples must be
pipetted when using 4-capillary analysers. If less than four samples are analysed, fill
up the empty positions on the plate with 12 μL Hi-Di™ Formamide.
One allelic ladder should be run per capillary in order to ensure reliable allelic
assignment on 4-capillary analysers.
Room temperature can influence the performance of PCR products on multi-capillary
units, so split peaks may occur especially at low temperatures. Pay attention to
keeping ambient conditions as recommended by the instrument manufacturer.
Signal intensities
Options to increase the signal intensity:
- Reduce the volume of the DNA Size Standard 550 (ROX) to peak heights of about
500 relative fluorescent units (RFU)
- Purify the PCR products before starting the analysis
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3.3 Setting up the GeneMapper™ ID software
Edit the Run Module as follows for the first run:
- In the Module Manager of the Data Collection software click on New to open the
Run Module Editor dialog box
Run Module 20min_400bp
Parameter
Oven Temperature [°C]
Poly Fill Volume
Current Stability [μA]
PreRun Voltage [kV]
PreRun Time [s]
Injection Voltage [kV]
Injection Time [s]*
Voltage Number of Steps
Voltage Step Interval
Data Delay Time [s]
Run Voltage [kV]
Run Time [s]**
Value
60
4840
5
15
180
3
5
40
15
1
15.0
1200
* Deviating from the standard settings, the injection time may range between 1 and 20 s depending on the type
of sample. If samples with very high signal intensities are recorded, a shorter injection time may be selected.
For samples with low DNA content an injection time of up to 20 s may be necessary.
** Depending on the analysis conditions the run time for Mentype ® Argus Y-MHQS was modified in order to be
able to analyse fragments with lengths of up to 400 bp.
- Click on Save As, enter the name of the new module (e.g. 20min_400bp) and confirm with
OK
- Click on Close to exit the Run Module Editor
Starting the run
- Place the prepared 96-well plate on the autosampler tray
- In the Protocol Manager of the Data Collection software, click on New in the
Instrument Protocol window to open the Protocol Editor dialog box
Instrument Protocol
Protocol Editor
Name
Type
Run Module*
Dye Set
e.g. Run36_POP4_DS-30
REGULAR
HIDFragmentAnalysis36_POP4_1
D
* parameter see above
- Click on OK to exit the Protocol Editor
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Prior to each run, it is necessary to create a plate definition as follows:
- In the Plate Manager of the Data Collection software click on New to open the New
Plate Dialog box
GeneMapper™ Plate Editor (I)
New Plate Dialog
Name
Application
Plate Type
Owner Name / Operator Name
e.g. Plate_DS-30_Date
select GeneMapper Application
96-Well
…
- Click OK. A new table in the Pate Editor opens automatically
GeneMapper™ Plate Editor (II)
Column
Sample Name
Priority
Sample Type
Size Standard
Panel
Analysis Method
Snp Set
User-defined 1-3
Results Group 1
Instrument Protocol 1
Type name for the samples
e.g. 100 (Default)
Sample or Allelic Ladder
e.g. SST-ROX_50-400bp
e.g. Biotype_Panels_v2 (choose test kit)
e.g. Analysis_HID_3130
(select results group)
Run36_POP4_DS-30 (setting described earlier)
- Click into the column header to select the entire column, select Edit → Fill Down to
apply the information to all selected samples and click on OK
- In the Run Scheduler, click on Find All, select Link to link the reaction plate on the
autosampler up with the newly created plate record (position A or B) and start the run
- During the run, view Error Status in the Event Log or examine the quality of the raw
data for each capillary in the Capillaries Viewer or the Cap/Array Viewer
- View data as overview in Run History or Cap/Array Viewer of the Data Collection
software. Run data are saved in the Run Folder of the previously chosen Result
Group
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3.4 Analysis parameter / analysis method
The recommended settings in the worksheet Peak Detector are:
Peak Detection Algorithm
Ranges
Smoothing and Baselining
Size Calling Method
Peak Detection
Advanced
Analysis: Partial Range
Start Pt: 2000; Stop Pt: 10000
Sizing: All Sizes
Smoothing: Light
Baseline Window: 51 pts
Local Southern Method
Peak Amplitude Thresholds
B:* Y:*
G:* R:*
Min. Peak Half Width: 2 pts
Polynominal Degree: 3
Peak Window Size: 11 pts**
Slope Thresholds: 0.0
* The peak amplitude threshold (cutoff value) corresponds to the minimum peak height that will be detected by
the GeneMapper™ ID software. The thresholds are usually 50-200 RFU and should be determined individually
by the laboratory. Recommendation: The minimal peak height should be three times as high as the background
noise of the baseline.
** Point alleles (i.e. alleles with at least 1 bp difference to the next integer allele) may occasionally not be
distinguished. For improved peak detection, minimise the Peak Window Size further.
Mentype® Argus Y-MHQS
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4. Analysis
For general instructions on automatic sample analysing, refer to the GeneScan® or
GeneMapper™ ID Software User’s Manual.
Finding the exact lengths of the amplified products depends on the device type, the
conditions of electrophoresis, as well as the DNA size standard used. Due to the
complexity of some loci, determining the size should be based on evenly distributed
references. The DNA Size Standard 550 (ROX) shall thus be used with the following
lengths of fragments: 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200, 220,
240, 260, 280, 300, 320, 340, 360, 380, 400, 425, 450, 475, 500, 525 and 550
bp.
Fig. 6 Electropherogram of the DNA Size Standard 550 (ROX), fragments with lengths in bp
Note: The basic template files for the DNA Size Standard 550 (ROX) has to be adjusted
to 400 bp within the GeneMapper™ ID software. The new template could be saved as
e.g. SST-ROX_50-400bp and used for further analyses.
As mentioned before, Mentype® Argus Y-MHQS contains an internal PCR control
(Quality Sensor), which provides helpful information on the efficiency of the PCR and on
presence of PCR inhibitors. A 6-FAM labelled 81 bp-fragment is amplified
independently of the DNA. The PCR control assay without DNA shows only the sensor
fragment (Fig. 7) and indicates successful polymerase chain reaction.
Fig. 7 Electropherogram of the 6-FAM labelled PCR control fragment (Quality Sensor). Fragment length in bp,
signal intensities in peak height
Mentype® Argus Y-MHQS
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Special features
In general, the electropherogram displays a single peak for each Y-STR locus.
However, locus DYS385 produces two peaks of different or same length. These two
fragments originate from duplicated and inversed copies of one Y-chromosomal locus.
The primers provided in the test kit simultaneously co-amplify the two homologous loci.
For separate amplification, see Kittler et al., 2003.
Concerning locus DYS385 it must be stressed that alleles 14.3, 15.3, 16.3, 17.3, and
19.3 represent the alleles 15, 16, 17, 18, and 20, respectively, with one thymidine
deletion between the primer binding site and the repeat region (Füredi et al., 1999).
This deletion may serve as an additional distinctive feature for differentiation in forensic
casework.
If more than one peak is obtained in the electropherogram for one or several markers,
this does not necessarily hint at mixed samples. Duplications or triplications of STR
markers also result in such an effect and have already been observed for DYS385 and
DYS19 (Butler et al., 2005). Rarely, single systems can fail too because of Ychromosomal deletions as known in azoospermic patients, as already described for
DYS385 and DYS392 (Stein et al., 2005).
Mutation rates for intermediate or infrequent alleles of the Y-STR system of the
Mentype® Argus Y-MHQS test kit varies between 0,613×10-3 and 5,296×10-3
(Gusmão et al., 2005b).
Allele frequencies of the European Minimal Haplotype Standard are collected and
published by the Biotype AG (Rodig et al., 2007). They can be received as download
from our homepage (www.biotype.de). Furthermore, there is free access to the “YChromosome Haplotype Reference Database” from the International Forensic Y-User
Group (http://www.yhrd.org/index.html) in order to calculate frequencies oneself.
Mentype® Argus Y-MHQS
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4.1 Biotype® template files
Allele allocation should be carried out with a suitable analysis software, e.g.
GeneMapper™ ID or Genotyper® software in combination with the Mentype®
Argus Y-MHQS template files from Biotype AG. Template files are available from our
homepage or as CD-ROM on request.
Recommended Biotype® templates for GeneMapper™ ID software are:
Panels
BinSets
Size Standard
Analysis Method
Plot Settings
Table Settings
Biotype_Panels_v2 (choose kit)
or higher versions
Biotype_Bins_v2
or higher versions
SST-BTO_50-500bp (adjust up to 400bp, adjustment described earlier)
Analysis_HID_310
Analysis_HID_3130
Plots_Blue
Plots_Green
Plots_Yellow
Plots_Red
Plots_4dyes
Table for 2 alleles
Table for 10 alleles
Panels and BinSets always have to be used whereas the other template files are
optional.
Recommended Biotype® template files for Genotyper® software are:
Argus_YMH-QS_v2c
or higher versions
General procedure for the analysis
1. Check the DNA size standard
2. Check the allelic ladder
3. Check the positive control
4. Check the negative control
5. Analyse and interpret the sample data
Mentype® Argus Y-MHQS
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4.2 Controls
The Control DNA XY1 of the test kit and other commercially available DNA from
standard cell lines represent the following alleles:
Table 3. Allele determinations of Mentype® Argus Y-MHQS
STR Locus
DYS19
DYS385ab
DYS389-I
DYS389-II
DYS390
DYS391
DYS392
DYS393
Control DNA
XY1
13
16 / 17
13
30
24
11
11
13
Control DNA
XX28
-
ATCC
K-562
-
CCR
9947
-
CCR
9948
14
11 / 14
13
31
24
10
13
13
CCR
3657
13
16 / 19
12
29
24
10
11
13
No PCR fragments are amplified with the Control DNA XX28, but may be used to
generate mixtures with the Control DNA XY1. The reference DNA K-562 is availble
from ATCC (http://www.atcc.org/Products/ PurifiedDNA.cfm#celllines). For further
confirmation, the table above displays the Y-chromosomal alleles of two reference
DNAs purchased from Coriell Cell Repositories (CCR; http://locus.umdnj.edu/nigms)
that is up to the standard of Szibor et al., 2003.
4.3 Lengths of fragments and alleles
Table 4 to table 5 show the fragment lengths of individual alleles that refer to the
DNA Size Standard 550 (BTO). All analyses have been performed on an
ABI PRISM® 310/3130 Genetic Analyzer with POP-4 polymer. Different analysis
instruments, DNA size standards or polymers may result in different fragment lengths.
In addition, a visual alignment with the allelic ladder is recommended.
Scaling
Horizontal: 85-405 bp (with Quality Sensor 75-405 bp)
Vertical: Depending on signal intensity
Mentype® Argus Y-MHQS
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Figure 8
A
QS
DYS393
DYS391
DYS390
DYS19
DYS385
DYS389-I
DYS392
DYS389-II
DNA Size Standard 550 (ROX)
B
QS
DYS393
DYS391
DYS390
DYS19
DYS385
DYS389-I
DYS392
DYS389-II
DNA Size Standard 550 (ROX)
Fig. 8 Electropherogram of the Mentype® Argus Y-MHQS using 500 pg Control DNA XY1 (A) or 100 pg male Control DNA XY1 mixed
with 100 ng female Control DNA XX28 (B). The Quality Sensor (QS) is shown at 81 bp. Analysis was performed on an ABI PRISM®
310 Genetic Analyzer with the DNA Size Standard 550 (ROX). Allele assignment was performed using the Genotyper® software and the
Mentype® Argus Y-MHQS template file.
Mentype® Argus Y-MHQS
December 2008
QS
Fig. 9 Electropherogram of the allelic ladder Mentype® Argus Y-MH
QS
software and the Mentype® Argus Y-MH template files.
.
analysed on an ABI PRISM® 310 Genetic Analyzer. Allele assignment was performed using the Genotyper®
25
Figure 9
Mentype® Argus Y-MHQS
December 2008
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Table 4. Fragment lengths of the allelic ladder Mentype® Argus Y-MHQS analysed
on an ABI PRISM® 310 Genetic Analyzer (blue panel)
Marker/allele Size [bp]*
Further
Allele**
Marker/allele Size [bp]*
PCR control
6-FAM
DYS390
6-FAM
Quality Sensor
81
20
201
21
205
Further
Allele**
Marker/allele Size [bp]*
Further
Allele**
DYS385
6-FAM
9
274
7, 8
10
278
10.1
17, 18, 19
DYS393
6-FAM
22
209
11
282
11
113
8, 9, 10
23
213
12
286
12
117
12.2
24
217
24.3
13
290
13
121
25
221
25.2
13.2
292
14
126
26
225
14
294
15
130
27
230
28, 29
15
298
16
134
15.2
300
15.3
16
302
16.2, 16.3
17
305
17.1, 17.2, 17.3
18
309
18.1
19
313
20
317
21
321
17, 18
Mentype® Argus Y-MHQS
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12.2
14.2, 14.3
22, 23, 24, 28
27
Table 5. Fragment lengths of the allelic ladder Mentype® Argus Y-MHQS analysed on an
ABI PRISM® 310 Genetic Analyzer (green panel)
Marker/allele Size [bp]*
DYS391
Further
Allele**
HEX
Marker/allele Size [bp]*
DYS389-I
Further
Allele**
HEX
Marker/allele Size [bp]*
DYS389-II
8, 9, 10, 10.2
HEX
8
106
6, 7
11
245
27
363
9
110
9.3
12
249
28
367
10
114
13
253
29
371
11
119
14
257
30
375
12
123
15
261
31
379
13
127
32
383
33
387
DYS19
HEX
11
178
12
182
13
186
14
191
15
16
17
203
18
207
16, 17
14
10
DYS392
HEX
7
292
10
301
11
304
12
307
13
310
195
14
313
199
15
316
14.3
Further
Allele**
23, 24, 25, 26
34, 35
6, 8, 9
11.1
16
19
* rounded to integer
** The “off-ladder” alleles of Biotype’s DNA pool are allocated with the actual Biotype® template files for GeneMapper™ ID or
Genotyper® software. For further alleles see amongst others http://www.cstl.nist.gov/biotech/strbase/str_fact.htm or
http://www.yhrd.org
Mentype® Argus Y-MHQS
December 2008
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5. Interpretation of results
As mentioned above, post PCR analysis and automatic allele assignment with
suitable analysis software ensure a precise and reliable discrimination of
alleles.
Pull-up peaks
Pull-up peaks may occur if peak heights are outside the linear detection range
(>3000 RFU), or if an incorrect matrix was applied. They appear at positions of
specific peaks in other colour channels, typically with lower signal intensities.
Peak heights should not exceed 3000 RFU in order to prevent pull-up peaks.
Stutter peaks
The occurrence of stutter peaks depends on the sequence of the repeat
structure and the number of alleles. n-4 peaks are caused by a loss of a repeat
unit during amplification of tetranucleotide STR motives, caused by slippage
effects of the Taq DNA Polymerase, whereas n+3 and n-3 peaks appear
particularly during amplification of the trinucleotide STR motif DYS392 (Mulero
et al, 2006). Those peaks should be interpreted in accordance with the
template files of the Genotyper® and GeneMapper™ ID software.
Template-independent addition of nucleotides
Because of its terminal transferase activity, the Taq DNA Polymerase tends to
add an adenosine radical at the 3’-end of the amplified DNA fragments. The
artefact peak is one base shorter than expected (-1 peaks). All Biotype®
primers are designed to minimise these artefacts. Artefact formation is further
reduced by the final extension step of the PCR protocol at 68°C for 60
minutes. Peak height of the artefact correlates with the amount of DNA.
Laboratories should define their own limits for analysis of the peaks.
Quality Sensor to check the PCR results
Mentype® Argus Y-MHQS contains an internal PCR check (Quality Sensor),
which provides helpful information on the efficiency of the PCR and on
presence of PCR inhibitors (see Fig. 7). Complete sensor failure indicates total
inhibition of the PCR or errors in the assay. If the sensor signal is amplified in
presence of DNA either in the negative control or in the positive control, the
PCR is not inhibited. Samples with sufficient DNA and without inhibiting
substances result in the DNA profile according to the kit and the sensor
fragment. Reduced sensor peak heights in forensic samples indicate partial
PCR inhibition. If only the Quality Sensor is amplified, the sample contains very
little, only female or degraded DNA.
Mentype® Argus Y-MHQS
December 2008
29
References
Butler JM, Decker AE, Kline MC, Vallone PM (2005) Chromosomal
duplications along the Y-chromosome and their potential impact on Y-STR
interpretation. J. Forensic Sci. 50(4): 1-7.
Füredi S, Woller J, Padar Z, Angyal M (1999) Y-STR haplotyping in two
Hungarian populations. Int. J. Legal Med. 113(1): 38-42.
Gill P, Brenner C, Brinkmann B, Budowle B, Carracedo A, Jobling MA,
de Knijff P, Kayser M, Krawczak M, Mayr WR, Morling N, Olaisen B,
Pascali V, Prinz M, Roewer L, Schneider PM, Sajantila A, Tyler-Smith C
(2001a) DNA commission of the International Society of Forensic Genetics:
recommendations on forensic analysis using Y-chromosome STRs. Int. J. Legal
Med. 114(6): 305-9.
Gill P, Brenner C, Brinkmann B, Budowle B, Carracedo A, Jobling MA,
de Knijff P, Kayser M, Krawczak M, Mayr WR, Morling N, Olaisen B,
Pascali V, Prinz M, Roewer L, Schneider PM, Sajantila A, Tyler-Smith C
(2001b) DNA Commission of the International Society of Forensic Genetics:
recommendations on forensic analysis using Y-chromosome STRs. Forensic
Sci. Int. 124(1): 5-10.
Gusmão L, Butler JM, Carracedo A, Gill P, Kayser M, Mayr WR, Morling
N, Prinz M, Roewer L, Tyler-Smith C, Schneider PM (2005a) DNA
Commission of the International Society of Forensic Genetics (ISFG): an update
of the recommendations on the use of Y-STRs in forensic analysis. Int. J. Legal
Med. 119: 1-10.
Gusmão L, Sanchez-Diz P, Calafell F, Martin P, Alonso CA, AlvarezFernandez F, Alves C, Borjas-Fajardo L, Bozzo WR, Bravo ML, Builes
JJ, Capilla J, Carvalho M, Castillo C, Catanesi CI, Corach D, Di Lonardo
AM, Espinheira R, Fagundes de Carvalho E, Farfan MJ, Figueiredo HP,
Gomes I, Lojo MM, Marino M, Pinheiro MF, Pontes ML, Prieto V,
Ramos-Luis E, Riancho JA, Souza Goes AC, Santapa OA, Sumita DR,
Vallejo G, Vidal Rioja L, Vide MC, Vieira da Silva CI, Whittle MR, Zabala
W, Zarrabeitia MT, Alonso A, Carracedo A, Amorim A (2005b) Mutation
rates at Y chromosome specific microsatellites. Hum Mutat. 26(6):520-8.
Kittler R, Erler A, Brauer S, Stoneking M, Kayser M (2003) Apparent
intrachromosomal exchange on the human Y chromosome explained by
population history. Eur. J. Hum. Genet. 11(4): 304-14.
Mulero JJ, Chang CW, Hennessy LK (2006) Characterization of the N+3
Stutter Product in the Trinucleotide Repeat Locus DYS392. J. Forensic Sci.
51(5): 1069-1073.
Mentype® Argus Y-MHQS
December 2008
30
Rodig H, Grum M, Grimmecke HD (2007) Population study and evaluation
of 20 Y-chromosome STR loci in Germans. Int. J. Legal Med 121(1): 24-27.
Stein B, Willuweit S, Nagy M, Vogt PH, Roewer L (2005) AZF deletions of
the Y chromosome and failed amplification of commonly used Y-STRs. 21st
Congress of the International Society for Forensic Genetics. Ponta Delgada,
Portugal.
Szibor R, Edelmann J, Hering S, Plate I, Wittig H, Roewer L, Wiegand P,
Calì F, Romano V, Michael M (2003) Cell line DNA typing in forensic
genetics – the necessity of reliable standards. Forensic Sci. Int. 138 37-43.
Mentype® Argus Y-MHQS
December 2008
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Notes
Mentype® Argus Y-MHQS
December 2008
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Notes
Mentype® Argus Y-MHQS
December 2008
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