Polymerase Chain Reaction Chromosome 16: PV92Alu PCR TM

PCR History Greatly increases the quantity of a specific DNA sequence from as little as one molecule of DNA or RNA.Greatly increases the quantity of a specific DNA sequence from as little as one molecule of DNA or RNA. First developed in 1986 by Kary Mullis, who later received the Nobel Prize for his discovery. First developed in 1986 by Kary Mullis, who later received the Nobel Prize for his discovery. Used today in recombinant DNA, DNA sequencing, evolution and forensics. Used today in recombinant DNA, DNA sequencing, evolution and forensics.

Introduction 1. Extract genomic DNA 2. Prepare PCR samples & run 3. Gel electrophoresis

Extracting Genomic DNA Genomic DNA will come from buccal epithelial cells Rinse vigorously with 0.9% NaCl (i.e., normal saline solution) to loosen cheek cells Spin 1.5ml of cell suspension to pellet cells out from normal saline Repeat 2 times

Remove excess supernatant Resuspend cells in residual NS by pipette mixing Transfer the resuspended cells to a clean microfuge tube & add Insta-gene matrix Insta-gene matrix – a suspension of negatively charged beads that bind divalent cations to prevent DNA degradation Extracting Genomic DNA

Heat to 56˚C to break up connective tissue elements & denature DNAases Heat to 100˚C to lyse cells & release DNA Vortex during heating to mix Insta-gene matrix beads and cells After heating, centrifuge to remove Insta-gene beads DNA is now in supernatant Extracting Genomic DNA

Carefully remove DNA using pipette to aspirate supernatant Do not aspirate any of the matrix beads. They chelate divalent cations – if they are transferred to the next step, they will inhibit the PCR reaction from occurring. Extracting Genomic DNA Insta-gene matrix beads

1)Activated Precursors – deoxynucleotides dNTPs (dATP, dGTP, dCTP, & dTTP) 2) DNA Polymerase (Taq polymerase) 3) preexiting RNA molecule (primer) 4) template DNA 5) optimum conditions (pH, temperature, etc.) PCR Requirements

1)Activated Precursors – deoxynucleotides dNTPs (dATP, dGTP, dCTP, & dTTP) 2) Taq polymerase 3) preexiting RNA molecule (primer) 4) template DNA 5) optimum conditions (pH, temperature, etc.) PCR Reaction

Thermocycling Heat (94 o C) to denature DNA strands. Cool (58 o C) to anneal primers to template. Warm (72 o C) to activate Taq polymerase. Taq extends primers and replicates DNA. Repeat multiple cycles.

Heating causes DNA strands to separate Denature DNA strands 94 o C (30 seconds) (30 seconds) 5’ 3’ 5’ 3’ 5’ 3’ 5’

Annealing Primers Primers bind to the template sequence Taq Polymerase recognizes double- stranded substrate 5’ Primers anneal 58 o C (30 seconds) (30 seconds) 3’ 5’ 3’

Taq Polymerase extends primer DNA is replicated Extend 72 o C (60 seconds) Repeat denaturing, annealing, & extending cycles. 3’ 5’ 3’

Alu Amplified Region 400 bps 50 bps 300 bps Amplified Region 100 bps Homozygous 400/400 Heterozygous 400/100 Homozygous 100/100 Alu Insertion in Intron

Actual Alu-PCR Results Source: DNA Learning Center, Cold Spring Harbor Laboratory, 1994 PV92 Locus on Chromosome bp 641 bp 941 bp 641 bp