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Lab 8: PCR (Polymerase Chain Reaction)

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1 Lab 8: PCR (Polymerase Chain Reaction)
By Kristi Schramm Gabrielino HS

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. In criminal forensics, PCR is used to amplify DNA evidence from small samples that may have been left at a crime scene. PCR can be used to amplify DNA for genetic disease screening

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

4 Lab 8: PCR (Polymerase Chain Reaction)

5 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.

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

7 Quick Review on Exons and Introns

8 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.

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

10 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.

11 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

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

13 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?

14 What do we use? Reagents and supplies Equipment 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 1.85 µL ddH2O Master mix II (10 µL/reaction) 0.75 µL MgCl2 (50 mM) 9.25 µL ddH2O

15 Expected Results of PCR
Marker Homozygous Alu + Homozygous Alu – Heterozygous

16 Expected Results


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

19 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

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