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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, MgCl 2 , 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 ~95C used to denature the template DNA; step 2 is performed at a temperature between 37C and 65C (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.

First lab Meeting

  • 1 • Isolate genomic DNA from your cheek cells

  • 2 run

up

Set

and

a

PCR

reaction

on your DNA using D1S80 primers with HotStar

Taq

Polymerase*.

  • 3 run

Set

up and

a PCR

reaction

on your genomic DNA using the ACE primers

with Taq

Polymerase.

  • 4 • 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.

  • 5 [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.]

  • 6 • 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

  • 1 • 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.

  • 2 • 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.

  • 3 • Answer any questions about the format of your lab reports

  • 4 • 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.

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 100C 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 Mg 2+ , 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.

PCR mix preparations

Hotstar PCR with D1S80 primers (for VNTR )

 

1Rx( l)

5Rx( l) 225
5Rx( l)
225

10Rx( l)

15Rx( l ) 675
15Rx( l
)
675

20Rx( l)

Negative Control ( l) 45 5 l water
Negative
Control ( l)
45
5 l water
 

Hotstar PCR mix

45

  • 450 900

Template

5

Cycles

95C 15min; then 35 cycles of (94C 1min, 65C 1 min, 72C 1min); followed by 72C 10min; then hold at 4C.

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)

 

1Rx( l)

5Rx( l)

10Rx( l)

15Rx( l)

20Rx( l)

Negative Control ( l)

PCR master mix

25

125

250

375

500

25

Primer mix

2

10

20

30

40

2

Water

18

90

  • 180 360

270

18

Total

45

225

  • 450 900

675

45

Template

5

5 l water

Cycles

94C 3min; then 35cycles of (94C 1min, 58C 1 min, 72C 1min); followed by 72C 10 min; then hold at 4C.

Note: Taq PCR master mix (from Qiagen) contains PCR buffer, Mg 2+ , dNTP and Taq DNA polymerase.

 
 

PCR with GammaSTR ® Fluorescent STR System (four loci)

 

1Rx( l )

5Rx( l) 112.5
5Rx( l)
112.5

10Rx( l)

15Rx( l ) 337.5
15Rx( l
)
337.5

20Rx( l)

Negative Control ( l) 22.5 2.5 l water
Negative
Control ( l)
22.5
2.5 l water

PCR mix

22.5

  • 225 450

Template(1ng DNA)

2.5

Cycles

96C 1min; then 10 cycles of (94C 30sec, 60C 30sec, 70C 45sec); then 20 cycles of (90C 30sec, 60C 30sec, 70C 45sec); followed by 60C 30min; then hold at 4C(See protocol for ramp conditions). Store the PCR product at -20C.

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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