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 ChelexBoil (lyse cells and destroy nuclease)CentrifugeExtract DNA sample
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.
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 elementthe 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 strandsCool (56oC) to anneal primers to templateWarm (72oC) to activate Taq polymerase, which extends primers and replicates DNARepeat 40 cycles
12 PCR 94 C: Denature DNA 56 C: Anneal Primers to Template 72 C: Activates Taq PolymeraseRepeats 31 times
13 The PCR Reaction What do you need? What is needed for PCR?Template - the DNA to be amplifiedPrimers - 2 short specific pieces of DNA whose sequence flanks the target sequenceForwardReverseNucleotides - dATP, dCTP, dGTP, dTTPMagnesium chloride - enzyme cofactorBuffer - maintains pH & contains saltTaq 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 tipsMaster mix I (10 µL/reaction) Thermal cycler2.5 µL 10x PCR buffer w/o MgCl20.5 µL dNTP’s (10 mM)2.5 µL Forward primer (4pM/ µL)2.5 µL Reverse primer (4pM/ µL)0.15 µL Taq polymerase1.85 µL ddH2OMaster mix II (10 µL/reaction)0.75 µL MgCl2 (50 mM)9.25 µL ddH2O
15 Expected Results of PCR MarkerHomozygous Alu +Homozygous Alu –Heterozygous
18 Huntington Disease Trinucleotide repeats (CAGCAGCAG…) Over 40 of these repeats causes the diseasePCR 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