EduPrimer™ DNA Profiling Kit

EduPrimer™ DNA Profiling Kit
GenoSensor Corporation
EduPrimer™ DNA Profiling Kit
Catalog #’s 3001-3002
Version C
September 2012
User Manual
EduPrimer™ DNA Profiling Kit
Table of Contents
Note for Instructors .......................................................................................................... 2
Kit Storage and Safety Instruction ................................................................................... 3
EduPrimer™ DNA Profiling Protocol ............................................................................... 4
Kit Components and Additional Required Materials ........................................................ 4
EduPrimer™ DNA Profiling Kit: Protocol ......................................................................... 5
EduPrimer™ DNA Profiling Kit: Technical Manual ........................................................ 10
Troubleshooting ............................................................................................................ 15
Technical Service .......................................................................................................... 16
Literature Citation
When describing a procedure for publication using these products, we would appreciate that
you refer to them as the EduPrimer™ DNA Profiling Kit.
Trademarks
EduPrimer™ is a trademark of GenoSensor.
1
Notes for Instructors
Kit Components and Storage Conditions:
Component
Solution A
Solution B
Cotton Swabs
2X PCR Master Mix
Positive Control DNA (Heterozygous)
Negative Control (DNase- RNase-free H2O)
DNA ladder
Storage
Room temp.
Room temp.
Room temp.
-20ºC
-20ºC
-20ºC
-20ºC
Preparation for DNA isolation and PCR
Set heat block or water bath to 95ºC
Thaw 2x PCR Master Mix on ice. Before opening tube, spin 10 sec at 6,000 rpm or greater
in microcentrifuge. Vortex 10 seconds, then spin again for 10 seconds.
Each package contains enough PCR 2X Master Mix for 30 reactions. Use 10 ul of 2X PCR
Master Mix with 10 ul template for a final PCR volume of 20 ul.
Up to 3 positive control materials are available for PCR reactions (10 ul from Positive Control
tube + 10 ul 2X PCR Master Mix for one reaction).
Run 1 or up to 3 negative control reactions.
Electrophoresis
Electrophoresis reagents are not provided from the kit. Please refer required materials.
Best results are obtained by adding DNA dye (i.e. Gel Red or Sybr Safe) to molten agarose.
Avoid agarose gel from light. You can store and run the gel in a dark room, or cover the gel with
a box during gel polymerization and the whole electrophoresis process.
There is enough DNA ladder to load 3 lanes with 10 ul.
After PCR, load 10 ul of positive control reaction each lane.
After PCR, load 10 ul of negative control reaction each lane.
After PCR, load as much as 20 ul of student PCR reaction into a lane.
2
Shipping and Storage
EduPrimer™ DNA Profiling kits are shipped on blue ice. Components should be stored
at temperatures shown in the above table. At proper storage conditions, components are stable
for 1 year from the date received. Expiration dates are also noted on product labels.
Safety Warnings and Precautions
This product is intended for research use only. It is not recommended or intended for the
diagnosis of disease in humans or animals. Do not use internally or externally in humans or
animals. Consider all chemicals as potentially hazardous. Only persons trained in laboratory
techniques and familiar with the principles of good laboratory practice should handle these
products. Wear suitable protective clothing such as laboratory overalls, safety glasses, and
gloves. Exercise caution to avoid contact with skin or eyes: if contact should occur, wash
immediately with water (Material Safety Data Sheet for products is available upon request).
3
EduPrimer™ DNA Profiling Kit
Overview
The EduPrimer™ DNA Profiling Kit introduces Polymerase Chain Reaction (PCR) techniques to
students, or anyone wishing to learn PCR and its uses. It contains all reagents necessary for
DNA isolation and PCR. PCR is an extremely important and valuable skill to have in
contemporary biological and related sciences. In this particular kit, the experiment generates
varying results from person to person, demonstrating the basis for the process of creating DNA
profiles that are used to differentiate one person from another. After completing this
experiment, one should be able to proficiently perform PCR and understand the concepts
behind it.
Kit Components and Storage Conditions (for a lab of 24 students)
Component
Solution A
Solution B
Cotton Swabs
2X PCR Master Mix
Positive Control DNA
(Heterozygous)
Negative Control
Amount (30 rxn’s)
6 mL
0.6 mL
28
300 µL
30 µL
Storage
Room temp.
Room temp.
Room temp.
-20ºC
-20ºC
30 µL
30 µL
-20ºC
-20ºC
DNA ladder
Additional Required Materials














Thermal Cycler
Heat Block or (heat plate, Beaker with de-ionized water; water bath, Tube floater;
Thermometer)
Microcentrifuge
Microcentrifuge tubes
Vortex
Micropipettes (p10, p200, p1000)
Pipette tips
PCR tubes
Tube Racks
Ethanol or ethanol wipes
Electrophoresis equipment
Electrophoresis supplies: agarose, TBE, DNA loading buffer, running buffer, gel dye (eg.
Sybr safe, Gel Red)
Scissors and tweezers
UV light box or “Gel Doc” equipment and program
4
EduPrimer™ DNA Profiling Kit: Protocol
Preparation
1. Set heat block or water bath to 95ºC. For a heat block, it is recommended to add water
or sand to ensure proper heat transfer. For a water bath, be sure tubes are tightly sealed
and not fully submerged to avoid contamination.
2. Thaw 2x PCR Master Mix on ice. Before opening tube, spin 10 sec at 6,000 rpm or
greater in microcentrifuge. Vortex 10 seconds, then spin again for 10 seconds.
DNA Preparation
*Do not eat or brush teeth one hour prior to cheek cell collection. Wear gloves and handle
solutions carefully*
1. Add 200µl of Solution A (red label) to a marked microcentrifuge tube.
2. Collect cheek cells with provided swab. Thoroughly roll and swab inside cheek for 10
seconds.
3. Place swab into marked tube with Solution A.
4. Cut swab above tube to a length that will fit inside the tube. Make certain the cap will
shut tightly.
5. Press the tube against the vortex machine to thoroughly mix the sample, for at least 10
seconds. Solution A contains components which chemically disrupt cell membranes and
begin to unravel proteins. Under these conditions, the cheek cells will begin to lyse or
break open, spilling cell contents into the solution in the tube.
6. Place sample in heat block to incubate at 95°C for 5 minutes. Immediately place tube in
ice until ready for next step.This process continues to destroy proteins, particularly,
those that damage DNA.
7. Load sample into microcentrifuge (mini centrifuge works as well), taking care that there
is another sample directly across from your sample to keep the centrifuge in balance as
it spins. Close internal and external centrifuge lids.
8. Spin briefly (~10 seconds) to pool condensation that has collected on the cap.
9. Remove swab with tweezers (tweezers should be rinsed with ethanol between samples
to prevent contamination).
10. Add 20 µl Solution B (green label) to the sample tube. Solution B neutralizes the harsh
conditions needed for lysis, preparing the solution for DNA isolation and PCR to follow.
11. Close the tube lid tightly and vortex to mix for at least 10 seconds.
12. Load sample into microcentrifuge with the tube hinge pointing out, balancing out your
sample tube, and closing the lids.
13. Spin sample for 1 minute at 12,000 rpm.
14. Look for a small clear round pellet near the bottom of the tube under the hinge. This
pellet contains cellular debris. The aqueous solution (supernatant) that has not
precipitated into the pellet contains cellular DNA.
5
PCR Reaction Mixture
*Wear gloves and handle solutions carefully*
1. Prepare and label a small PCR tube with student’s name. Label both the top and side of
the PCR tube to ensure clarity.
2. Ensure that the “2x PCR Master Mix” is on ice and has been spun at 6,000 RPM or
greater in a microcentrifuge for 10 seconds, vortexed for 10 seconds, then spun again
for 10 seconds before opening the Master Mix tube.
3. (optional or based on instructor) Label and prepare PCR tubes for controls: 1-3 positive
and 1-3 negative. Follow below procedures, replacing student template DNA with
controls in their respective PCR tubes.
4. Add 10 µ l of “2X PCR Master Mix” and 10 µ l of supernatant (avoid the pellet) from
the 1.5 mL tube with the student “genomic DNA template” to the labeled PCR tube for at
total of 20 µ l as indicated in the table below. (Note: It is preferred that the PCR
reaction mix preparation is done on ice).
5. Mix the 20 µ l PCR reaction mixture by pipetting in and out with the pipette, and the
close the lid tightly.
6. Store the sample on ice until it is ready to be loaded into the thermal cycler.
PCR reaction mixture table
2x PCR Master Mix
10 µl
Genomic DNA Template
10 µl
____________________________________________
Volume total
20 µl
7. For positive and negative control tubes, below is the control PCR reaction mixture table.
Control PCR reaction mixture table
Positive control
Negative control
2x PCR Master Mix
10 µl
2x PCR Master Mix
10 µl
Positive control DNA
10 µl
Negative control
10 µl
_______________________________
_______________________________
Volume total
Volume total
20 µl
20 µl
6
Positive and negative controls give guidelines or boundaries to the experimental results.
Negative control show the result if no DNA is present for the PCR.
Positive control shows the result of PCR with purified DNA with a heterozygous genotype. PCR
products for the TPA-25 gene are 136 and 436 base pairs in length, for PV-92 180 and 480
base pairs.
DNA ladder is a standard which is used like a ruler to show how DNA of a certain length will
migrate through a gel during electrophoresis.
(+)- Each class will prepare 3 positive control reactions and 1 negative control reaction. Use 10
µl of provided positive control DNA (purple-red label) in 10 µl Master Mix (blue label.)
(-)-Substitute 10 µl of DNase free water (black label) for the template in a negative control PCR.
PCR Parameters
Program your thermal cycler as follows:
1.
2.
3.
4.
5.
6.
94ºC – 2 minutes
94ºC denaturing – 20 seconds}
58ºC annealing – 20 seconds} repeat steps 2, 3, & 4 for 40 cycles
72ºC extension – 20 seconds}
72ºC – 5 minutes
4ºC – finished / hold
Agarose Gel Electrophoresis
General Procedure, detailed directions as given by instructor

Pour 1% agarose gel

For staining, use a DNA dye which is added directly to the liquefied agarose.

Use at least10 µL of PCR product to visualize results by electrophoresis on agarose gel.
If gel well volume will accommodate more than 10 ul, a higher volume is preferred.
Loading dye is added to the sample to ensure that the sample will sink to the bottom of
the well and properly enter the agarose gel. Mix sample with loading dye according to
instructor directions before carefully pipetting into a well in the gel. Be sure to record
which well holds your sample.

Run at ~100V for ~20 minutes and stop before loading dye has run off gel. Depending
on the DNA dye used, caution may need to be taken to reduce exposure of gel to light.

Visualize under UV and record the results manually or by photography

Compare individual experimental bands to positive control DNA.
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Results and Discussion
The TPA-25 gene, as well as PV92, is in the Alu family of genes. Genes in this group may
contain a 300 base pair sequence that has inserted itself into an unexpressed (intron) portion of
the gene. This does not change the original flanking sequence of the gene so the same primers
will allow replication of either or both the longer or shorter version of the gene (see Figure
below).
8
Individuals may have inherited two copies of the gene with no insert and be homozygous. In
this case a single band on the gel at 136bp for TPA-25(Alu-), or 180bp for PV92 (Alu-) is
expected.
Individuals may have inherited two copies of the gene with the 300bp Alu insert and be
homozygous. In this case a single band on the gel at 436bp for TPA-25(Alu+), or 480bp for
PV92 (Alu+) is expected.
Individuals may have inherited one copy of the gene with no insert and one copy of the gene
with the 300bp Alu insert and be heterozygous. In this case two bands on the gel at 136bp and
436bp for TPA-25(Alu), or 180bp and 480bp for PV92 (Alu) is expected.
Data Analysis
Class Allele Frequency
The results from the whole class can be analyzed to determine the allele frequency within the
class. Record the results in the table below.
Observed Class Genotypes
Genotypes
# of individuals
#
alleles
of frequency
(+)/(+) homozygous w/
Alu
# of (+) alleles
.
Total (+) & (-) alleles
(+)/(-) heterozygous
(+)
(-)
(-)/(-)
homozygous
w/out Alu
# of (-) alleles
.
Total (+) & (-) alleles
Sum=
Sum=
class total
#
alleles
Sum = 1.00
of
Each person has two alleles, consequently, in calculating allele frequency, both copies are
taken into account.
To determine the frequency of the allele with the Alu insert, double the number of individuals
with (+)/(+) homozygous genotypes and add the number of individuals with (+)/(-) heterozygous
genotypes. This sum accounts for all the (+) alleles.
To determine the frequency of the allele without the Alu insert, double the number of individuals
with (-)/(-) homozygous genotypes and add the number of individuals with (+)/(-) heterozygous
genotypes. This sum accounts for all the (-) alleles. Divide this sum
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Scientists use Alu frequency to study human populations. To learn examples and compare the
frequencies measured in your class to other classes and populations around the world, visit the
“Allele Server” in Cold Spring Harbor’s Dolan DNA Learning Center at
www.bioservers.org/bioserver/.
EduPrimer™ DNA Profiling Kit: Technical Manual
Introduction to PCR
In 1983, during his time working at Cetus Corporation, Kary Mullis developed a
technique that has changed the field of genetics and biological science in general. This
revolutionary process was termed “polymerase chain reaction,” or PCR. He earned the Nobel
Prize in Chemistry in 1993 due to his innovation. His technique enabled researchers, besides a
few expert microbiologists, to amplify DNA. Before that, amplification of DNA was extremely
difficult and time consuming. Now, scientists in any field can incorporate molecular biology into
their research with PCR.
Currently, PCR is used in a wide variety of areas, for example, gene mapping, DNA
sequencing, gene expression, gene detection, forensics, criminal investigation, medical
diagnostics, and genome sequencing. Very few of these applications were practically possible
before PCR. The experiment does require an initial investment in specially made machinery,
but with the proper equipment, nearly anyone can perform a successful PCR experiment without
significant cost.
PCR and Biotechnology — Revolutionizes an Entire Research Community
PCR is capable of producing large amounts of targeted DNA from an extremely small
amount of starting material, known as your template. DNA can be obtained from any cell such
as blood cells, hair cells, cheek cells etc., and after proper treatment to isolate DNA, PCR can
be applied to create millions of copies of virtually any desired DNA sequence. That is one of the
most significant powers of PCR, specificity. Although you may put an entire genome worth of
DNA into a PCR, it amplifies the exact piece of DNA desired and leaves the rest out.
The basic components of PCR:
- Reaction Buffer
- DNA nucleotides (dNTP’s) of each adenine, guanine, thymine and cytosine
- DNA polymerase
- 2 DNA oligonucleotide primers
- Template DNA (starting material)
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PCR Makes Use of Two Basic Processes in Molecular Genetics
1. Complementary DNA strand hybridization
For DNA to be amplified, one must have a known sequence which flanks the gene of
interest upstream and downstream. These sequences are used to create what are known as
‘oligonucleotide primers,’ meaning a short ~20 base pair nucleotide sequence which is used as
a starting point for DNA replication. The primers are said to be complementary to their target
regions, so they will anneal (attach) to those regions specifically. Primers are required by DNA
polymerase because it cannot add nucleotides without a preexisting chain.
Complementary Strand Hybridization occurs when two different oligonucleotide primers
anneal to each of their respective complementary base pair sequences on the template. They
are designed specifically to anneal at opposite ends of opposite strands of the specific
sequence of DNA that is desired to be amplified.
2. DNA strand synthesis via DNA polymerase
In a PCR, a special type of DNA polymerase is used that is able to withstand the
temperature fluctuations required for thermal cycling. Most DNA polymerases cannot tolerate
the high temperatures and fluctuations from ~60ºC-94ºC. The breakthrough in PCR came with
the isolation of DNA polymerase from a thermophilic bacterium known as Thermus aquaticus.
This bacterial species lives in high temperature steam vents and therefore its DNA polymerase
evolved to withstand extremely high temperatures.
During PCR, DNA is synthesized and multiplies by 2 each cycle, thus the growth of DNA
copy # over the reaction is exponential. In theory, after 30 cycles there will be 230. This is over
a billion copies of DNA. Yielding this amount of DNA allows the possibility of visualization
through a variety of means. One of the most popular visualization methods is agarose gel
electrophoresis.
Genes and DNA
The human genome contains 23 pairs of chromosomes that contain a total of
thirty to fifty thousand genes, most of which generally code for proteins. However, those genes
only comprise about 5% of the genome, leaving 95% as so-called non-coding DNA. This
noncoding DNA is found not only between, but within genes, splitting them into segments. In
eukaryotes, non-coding DNA found within genes is known as introns. The sequences that do
code for proteins are called exons. In eukaryotes, genomic DNA is transcribed into RNA
molecules containing both introns and exons for a particular gene. While the RNA is still in the
nucleus (before being transported out of the nucleus), the introns (in = stay within the nucleus)
must be removed from the RNA while the exons (ex = exit the nucleus) are spliced together to
form the complete coding sequence which will soon be translated into the protein. This process
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is called RNA splicing. Some genes may contain a few introns, others may contain dozens.
Interestingly, it is the non-coding ‘junk’ DNA that is useful to us when considering a DNA profile
of an individual, instead of the DNA that actually codes for life.
As discussed, functional segments of genes (exons) code for proteins. Proteins are
molecules that carry out most cellular functions. Exon sequences are therefore very similar
among individuals, because even slight difference can change the function of the protein in a
potentially harmful way (many diseases are caused by mutated proteins). Introns, however,
often vary in size and number among individuals. Intron sequences are thought to be the result
of the differential accumulation of mutations throughout evolution that are silently passed to
descendants through the hereditary code. It is this difference in intron sequences that allows us
to determine human genetic diversity. The identification of these distinctive characteristics in
DNA represents the molecular basis for human identification and population genetics.
Throughout evolution, intron sequences have been the target of random insertions by short
repetitive interspersed elements, also known as SINEs. SINEs have become randomly inserted
within our introns over millions of years.
One such repetitive element is called the Alu element (Figure 1). The Alu element is a
DNA sequence about 300 base pairs long that is repeated; one copy at a time, almost 500,000
times within the human genome. The origin and function of such randomly repeated sequences
is not yet known. The Alu name comes from the Alu I restriction enzyme (enzymes that cut DNA
at specific sequences) recognition site that is found in this sequence. The following section
reviews the Alu element in more detail.
PCR Stages
The machinery required to perform PCR is known as a thermal cycler. The thermal
cycler enables the steps of PCR to be automated. The reaction involves a repetitive series of
cycles, each of which consists of template denaturation, primer annealing, and extension of the
annealed primer by Taq DNA polymerase. Before beginning DNA amplification, genomic DNA is
prepared from students' cells. The students’ DNA is then added to a mixture of the necessary
reagents: oligonucleotide primers, thermostable DNA polymerase (Taq), the four
deoxynucleotides (A, T, G, C), and reaction buffer. These reagents are pre-mixed as a 2X PCR
Master Mix in the EduPrimer™ DNA profiling kit. The tubes are placed into the thermal cycler.
These thermal cyclers contain an aluminum block that holds the samples and can be rapidly
heated and cooled across extreme temperature differences. The rapid heating and cooling of
this thermal block is called temperature cycling or thermal cycling.
The first step of the PCR temperature cycling procedure involves heating the sample to
94°C. At this high temperature, the template strands separate. This is called the denaturation
step.
The thermal cycler then rapidly cools to 60°C to allow the primers to anneal to the
separated template strands. This is called the annealing step. The two original template strands
12
may reanneal to each other or compete with the primers for the primers complementary binding
sites. However, the primers are added in excess such that the primers actually out-compete the
original DNA strands for the primers’ complementary binding sites.
Lastly, the thermal cycler heats the sample to 72°C for Taq DNA polymerase to extend
the primers and make complete copies of each template DNA strand. This is called the
extension step. Taq polymerase works most efficiently at this temperature. Two new copies of
each complementary strand are created. There are now two sets of double-stranded DNA
(dsDNA). These two sets of dsDNA can now be used for another cycle and subsequent strand
synthesis. At this stage, a complete temperature cycle (thermal cycle) has been completed.
Each step takes about 30 seconds to 1 minute, and this process continues for roughly
30-40 cycles depending on how the user has programmed the thermal cycler. Each step is
repeated in that order each cycle until it is completed. At the end, the product is put on hold at a
low temperature, generally 4°C, until the user is ready to proceed to the analysis of the product.
13
Figure 3. Experiment flowchart from start to finish
14
Troubleshooting
Symptom
Possible causes
Solutions
No amplification
product
Questionable template quality
Analyze starting material
Inhibitory Substance in reaction
Decrease sample volume
Insufficient cycle #
Run additional cycles
Incorrect thermal cycler program
Verify times and
temperatures
Errors in heat block incubation
Calibrate heating block, use
sand or water to maximize
contact with tube for proper
heat transfer
Contaminated tubes/solutions
Autoclave tubes and use
filter tips
Lower annealing
temperature in 2º
increments
Mix solutions on ice, place
rxn directly to 94º thermal
cycler
Raise annealing
temperature in 2º
increments
Mix solutions thoroughly
before beginning the
reaction
-Wear gloves
-Use dedicated area for
sample preparation
-Use non-aerosol tips
Primer annealing temperature too
high
Non-specific
amplification
product
Premature Taq-polymerase
replication
Primer annealing temperature too
low
Insufficient mixing of reaction
solution
Exogenous DNA contamination
15
Technical Service
For more information or technical assistance, please call, write, fax, or email.
GenoSensor Corporation
4665 S. Ash Avenue
Suite G-18
Tempe, Arizona 85282
Tel: 1-480-598-5378
Fax: 1-480-755-3319
Email: [email protected]
Web: www.genosensorcorp.com
Limited Warranty
GenoSensor is committed to providing our customers with high-quality goods and services. Our goal is to
ensure that every customer is 100% satisfied with our products and our service. If you should have any
questions or concerns about a GenoSensor product or service, please contact our Technical Service at
[email protected] GenoSensor warrants that all of its products will perform according
to the specifications stated on the certificate of analysis. This warranty limits GenoSensor Corporation’s
liability only to the cost of the product. No warranty is granted for products beyond their listed expiration
date. No warranty is applicable unless all product components are stored in accordance with instructions.
GenoSensor reserves the right to select the method(s) used to analyze a product unless GenoSensor
agrees to a specified method in writing prior to acceptance of the order. GenoSensor makes every effort
to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is
inevitable. Therefore GenoSensor makes no warranty of any kind regarding the contents of any
publications or documentation. If you discover an error in any of our publications, please report it to our
Technical Service. GenoSensor assumes no responsibility or liability for any special, incidental, indirect or
consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other
warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a
particular purpose
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