1. Lab 8: PCR (Polymerase Chain Reaction)

2. PCR – Polymerase Chain Reaction has many applications
PCR is commonly used to produce many copies of a selected gene segment or locus of DNA.

3. PCR – Polymerase Chain Reaction has many applications
In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene.

4. PCR – Polymerase Chain Reaction has many applications
PCR can be used to amplify DNA for genetic disease screening or paternity tests.

5. Lab 8: Obtaining DNA Sample
Add cheek cells to Chelex
Boil (lyse cells and destroy nuclease)
Centrifuge
Extract DNA sample

6. How Does PCR Work? Virtual PCR: http://learn.genetics.utah.edu/
To imbed video into ppt:
PCR Video

7. Marty’s PCR Video ..\..\Amgen-BW CD, 2006\PCR\Pcr.exe

8. Lab 8: PCR (Polymerase Chain Reaction)

9. The locus we will amplify is located in the tissue Plasminogen Activator (tPA) gene.
This gene is on chromosome 8.
The gene codes for a protein that is involved with dissolving blood clots.
tPA is a protein given to heart attack victims to reduce the incidence of strokes.

10. The region we will be amplifying is located in an intron (non-translated region), of the tPA gene.

11. Quick Review on Exons and Introns

12. The intron that we will be targeting for amplification is dimorphic, which means the locus has two forms.
one form carries a 300 bp DNA fragment known as an Alu element
the second form of the locus does not carry this fragment.

13. .
The diagram indicates the intron we will be targeting for PCR.
Two Possibilities: 100 bp sequence 400bp sequence

14. What are Alu elements?
Alu elements are short, around 300 bp, DNA fragments that are distributed throughout our genome.
Estimated that we may carry over 1,000,000 copies of this fragment.

15. Karyotype with Alu tagged
A probe with a florescent tag was created that was specific to the alu segment.
Chromosomes were ‘denatured’ and spread on a slide, then arranged.
Less than .5% are polymorphic

16. The PCR Reaction How does it work?
Heat (94oC) to denature DNA strands
Cool (56oC) to anneal primers to template
Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA
Repeat 40 cycles

17. PCR 94 C: Denature DNA 56 C: Anneal Primers to Template
72 C: Activates Taq Polymerase
Repeats 31 times

18. The PCR Reaction What do you need?
What is needed for PCR?
Template - the DNA to be amplified
Primers - 2 short specific pieces of DNA whose sequence flanks the target sequence
Forward
Reverse
Nucleotides - dATP, dCTP, dGTP, dTTP
Magnesium chloride - enzyme cofactor
Buffer - maintains pH & contains salt
Taq DNA polymerase – thermophillic enzyme from hot springs (Thermus aquaticus)
The PCR Reaction What do you need?

19. What do we use? Reagents and supplies
Genomic DNA sample (5 µL) P-20 pipette and tips
Master mix I (10 µL/reaction) Thermal cycler
2.5 µL 10x PCR buffer w/o MgCl2
0.5 µL dNTP’s (10 mM)
2.5 µL Forward primer (4pM/ µL)
2.5 µL Reverse primer (4pM/ µL)
0.15 µL Taq polymerase (comes in PCR tube, must be vortexed because it is very dense)
1.85 µL ddH2O
Master mix II (10 µL/reaction)
0.75 µL MgCl2 (50 mM)
9.25 µL ddH2O

20. Electrophoresis of PCR Sample:
Make a 2% agarose gel:
Calculations: 0.6 grams agarose in 30 mL 1X SB Buffer
Add 3µL of orange G your PCR tube containing your amplified DNA sample
Load into gel in numerical order
Run gel at 135 V until the Orange G has migrated just past halfway across the gel.
Stain gel with Ethidium Bromide for 10 min
De-stain with water for 10 min
Photograph gel
Analyze results (lab 8 conclusion questions)

21. Expected Results of PCR
Marker
Homozygous Alu +
Homozygous Alu –
Heterozygous

22. Expected Results

23. PCR Results
‘M’ A B C D F G H I K L M
‘M’ N O P Q R T W Z ‘mystery’

24. Huntington Disease Trinucleotide repeats (CAGCAGCAG…)
Over 40 of these repeats causes the disease
PCR can be used to identify this disease

25. The Alu element maybe a part of the DNA coding for an RNA molecule that aids in the secretion of newly formed polypeptides from the cell.
it has little if any effect on protein function unless it happens to become inserted into an exon or coding region