MCB 730: Polymerase Chain Reaction (PCR) DNA Fingerprinting Lab
Instructor: Dr Allan Showalter - Porter 504
Required reading: Bloom, M.V., Freyer, G.A. and Micklos, D.A. (1995) Laboratory DNA Science,
Laboratory 22 and 23, Benjamin/ Cummings Publishing Co., New York, pp. 343-371
Background: The polymerase chain reaction (PCR) is a powerful technique for the amplification of
small amounts of DNA. In general, this technique involves the addition of template (or target) DNA,
two oligonucleotide primers complementary to the template DNA, the four deoxynucleotides, a PCR
buffer, MgCl2, and a thermostable DNA polymerase (i.e.,Taq polymerase). This mixture then undergoes
repeated three-step cycles to amplify the template DNA located between the two oligonucleotide
primers. Step 1 is a high temperature incubation at ~95C used to denature the template DNA; step 2 is
performed at a temperature between 37C and 65C (note: the exact temperature depends upon the
length and sequence of the two primers) to allow for annealing of the primers to the denatured template
DNA; and step 3 is performed to extend the primer sequences and synthesize DNA complementary to
the template strands.
In this lab, you will learn how to detect a variable number tandem repeat (VNTR) and short tandem
repeats (STR) polymorphisms in humans by PCR and use this information to solve a heinous crime. You
will isolate your own DNA from your cheek cells and amplify a portion of your genomic DNA (D1S80
locus) corresponding to a VNTR sequence located on chromosome 1 using two primer sequences called
D1S80U and D1S80D. This VNTR is highly polymorphic in the human population and can provide the
basis for forensic identification as well as paternity testing. You will also amplify a portion of your
genomic DNA corresponding to the angiotensin converting enzyme (ACE) gene, which is polymorphic
in the human population with respect to the insertion of an Alu element (see handout for details). The
primer sequences that you will use, referred to a primer 1 and primer 2, are ACE-specific
oligonucleotide primers.
New this year, we will be using Promega’s GammaSTR® Fluorescent STR System. As stated by
Promega, this system “provides a rapid, non-radioactive method designed to obtain a DNA type, or
genetic fingerprint using very small amounts (e.g., 1ng) of human DNA. The four STR loci that are
included in this multiplex system, D16S539, D7S820, D13S317 and D5S818, contain alleles of discrete
and separable lengths. This allows the construction of allelic ladders that contain fragments of the same
length as several or all known alleles for the locus. Visual comparison between the allelic ladder and
amplified samples of the same locus allows rapid and precise assignment of alleles.”
Each student will work with his or her own DNA and is required to submit an individual lab report by
5:00pm Wednesday, May 2, 2007. Your lab report should contain the following: an introduction for
these PCR labs, a brief summary of materials and methods employed, the results of your PCR
experiments as well as that your lab-mates, discussion of all results, including the likely murderer as
well as possible ways in which you might incorporate PCR methodology into your own research, and a
list of cited references.
You will note that there are many variations and uses of the basic PCR methodology such as reverse
transcriptase PCR (RT PCR), differential display RT PCR, inverse PCR, genomic PCR, DNA
sequencing by PCR, diagnostic PCR, forensic PCR, and molecular evolutionary studies using PCR.
Note also that the RESULTS and DISCUSSSION section on page 369-371 of your handout should be
used as a guide in writing up your lab report.
1
First lab Meeting
• Isolate genomic DNA from your cheek cells
• Set up and run a PCR reaction on your DNA using D1S80 primers with HotStar Taq
Polymerase*.
• Set up and run a PCR reaction on your genomic DNA using the ACE primers with Taq
Polymerase.
• Set up and run a PCR reaction on your genomic DNA using one of Promega’s GenePrint ®
Fluorescent STR Systems: the GammaSTR® Multiplex System (Catolog #DC6071-$675) with
Taq Polymerase.
[Note The GammaSTR® Fluorescent STR System provides a rapid, non-radioactive method
designed to obtain a DNA type, or genetic fingerprint using very small amounts (e.g., 1ng) of
human DNA. The four STR loci that are included in this multiplex system, D16S539, D7S820,
D13S317 and D5S818, contain alleles of discrete and separable lengths. This allows the
construction of allelic ladders that contain fragments of the same length as several or all known
alleles for the locus. Visual comparison between the allelic ladder and amplified samples of the
same locus allows rapid and precise assignment of alleles.]
• The TA will pre-run the PCR samples to ensure everyone has PCR products.
* HotStarTaq polymerase is a modified form of the Taq polymerase. It is provided in an inactive state
with no polymerase activity at ambient temperatures. This prevents the formation of misprimed product
and primer-dimers at low temperature. HotStar Taq polymerase is activated by a 15 min, 95°C
incubation step, which can easily be incorporated into existing thermal cycling programs. HotStar Taq
polymerase provides high PCR specificity and often increases the yield of the specific PCR product.
Second lab Meeting
• Run your two (D1S80 and ACE) PCR samples on the two corresponding 2% Agarose gels (i.e.
2.4g of agarose in 120 ml of 1X TAE) containing ethidium bromide (i.e., 7μl of a 10mg/ml stock
solution) and then the TA will photograph the gel and post it on the course web page.
• Watch Vijay Nadella load/run your GammaSTR® Fluorescent STR PCR samples on the ABI
PRISM® 3130 Genetic Analyzer and then the TA will post the output on the course web page.
• Answer any questions about the format of your lab reports
• Discuss primer design and other uses of PCR
First lab meeting protocol
1. Print your name by your assigned number on the sheet provided
2. Pour all the 0.9% saline solution in the 15ml centrifuge tube assigned to you into your mouth and
vigorously swish for 15 seconds. Save empty tube for use in next step.
3. Expel the sample solution into a paper cup. Carefully pour the saline solution from the paper cup
back into the 15ml centrifuge tube from step 2.
4. Spin sample in preparatory centrifuge on high speed (500-1000Xg, usually 2000-3000rpm for most
tabletop clinical centrifuges) for 10 minutes.
2
5. Carefully pour off supernatant into sink and place tube containing your cells on ice.
6. Use the 1000l micropipettor to transfer 500l 10% Chelex solution to the tube containing your cell
pellet. (Set the micropippettor to 500l and pipet the Chelex solution up and down several times to
resuspend the Chelex beads before adding them to the centrifuge tube containing your cell pellet.)
7. Mix cells and Chelex by pipeting up and down several times until no visible clumps of cells remain.
8. Transfer 500 l of your resuspended sample into a clean 1.5ml tube. Make sure to label the cap of
the tube with your assigned number and punch a hole in the lid with a needle.
9. Place your tube in a 100C air bath for 15 minutes.
10. Carefully remove your tube from the boiling water bath, votex for 10 sec and then place on ice for 1
minute.
11. Centrifuge your tube for 30 sec in a microcentrifuge at the highest speed.
12. Use a fresh pipet tip to transfer 200l of the supernatant to another clean 1.5ml tube. Label with your
assigned number. Do not transfer any pellet since the Chelex will interact with Mg2+, which is
critical for the PCR reaction.
13. Set up the first two PCR reactions (D1S80 and Alu) as follows: Use a 200l tube to set up the two
PCR reactions: (5l cheek cell DNA + 45l of the appropriate PCR reaction mix)
14. Quantify your DNA sample using the Spectrophotometer, and dilute your DNA to a final
concentration of 1ng/2.5l.
15. Use a 200l tube to set up the third PCR reaction (STRs): (2.5l diluted cheek cell DNA +22.5l of
the appropriate PCR reaction mix). (See the details for the PCR reaction mixes on page 4.)
16. Place your tube in the appropriate thermal cycler (GeneAmp PCR system 9700). Run the PCR
according to the cycle conditions listed on page 4.
Second Lab meeting
One 120 ml 2% agarose gel containing ethidium bromide and 40 sample wells is prepared for you. The
gel will be electrophoresed in 1X TAE.
1. Each student will remove 10l of their PCR reaction and add it to a tube containing 2l of 6X
loading buffer. The sample should be mixed by pipeting the solution up and down a few times.
Label the tube with your assigned number.
2. Load your 12l sample in numerical order in the wells of the gel. A 100bp DNA ladder from
Promega will be used as the marker and will be added in the first and last wells. The gel will be
electrophoresed at 90 volts for approximately 2 hours during which time we will do RNA extraction
and RT-PCR and have a brief discussion of the lab reports and various PCR applications.
3. After electrophoresis is complete, the gels will be photographed and placed on the course web pag
4. While the gels are running, we will watch Vijay Nadella load/run your GammaSTR® Fluorescent
STR PCR samples on the ABI PRISM® 3130 Genetic Analyzer and then the TA will post the output
on the course web page.
5. While all your samples are running, we will then discuss the lab report and various PCR methods.
3
PCR mix preparations
Hotstar PCR with D1S80 primers (for VNTR )
Hotstar PCR mix
Template
Cycles
1Rx(l)
45
5
5Rx(l)
225
10Rx(l)
450
15Rx(l)
675
20Rx(l)
900
Negative
Control (l)
45
5l water
95C 15min; then 35 cycles of (94C 1min, 65C 1 min, 72C 1min); followed by
72C 10min; then hold at 4C.
Note: The Hotstar PCR mix (from Qiagen and made by TA) contains PCR buffer, Mg 2+, dNTP, primer mix,
and Hotstar Taq DNA polymerase.
Regular PCR with the ACE primers (for Alu polymorphism)
PCR master mix
Primer mix
Water
Total
Template
Cycles
1Rx(l)
25
2
18
45
5
5Rx(l)
125
10
90
225
10Rx(l)
250
20
180
450
15Rx(l)
375
30
270
675
Negative
20Rx(l) Control (l)
500
25
40
2
360
18
900
45
5l water
94C 3min; then 35cycles of (94C 1min, 58C 1 min, 72C 1min); followed by
72C 10 min; then hold at 4C.
Note: Taq PCR master mix (from Qiagen) contains PCR buffer, Mg2+, dNTP and Taq
DNA polymerase.
PCR with GammaSTR® Fluorescent STR System (four loci)
PCR mix
Template(1ng DNA)
Cycles
Negative
1Rx(l)
5Rx(l)
10Rx(l)
15Rx(l)
20Rx(l) Control (l)
22.5
112.5
225
337.5
450
22.5
2.5
2.5l water
96C 1min; then 10 cycles of (94C 30sec, 60C 30sec, 70C 45sec); then 20
cycles of (90C 30sec, 60C 30sec, 70C 45sec); followed by 60C 30min; then
hold at 4C(See protocol for ramp conditions). Store the PCR product at -20C.
Note: The PCR mix (from Promega and made by TA) contains STR buffer, multiplex primer pair mix,
and Taq DNA polymerase.
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