Polymerase Chain Reaction

PCR Repetitive amplification of a piece or region of DNA Numerous uses Straightforward amplification & cloning of DNA Mutagenesis Sequencing RT-PCR – reverse transcription coupled with PCR to amplify mRNAs (cDNAs actually are template) Production of cDNA libraries

PCR Requirements DNA template Oligodeoxynucleotide primers DNA that will be amplified (copied) Oligodeoxynucleotide primers anneal to template to allow DNA replication thermostable DNA polymerase DNA polymerase extends the primers to synthesize a copy of the template DNA thermostable polymerases allow automation and repeated rounds of DNA denaturation deoxynucleotides and appropriate reaction conditions dNTPs are incorporated into synthesized DNA, buffered pH, & Mg2+ to allow enzyme activity of DNA pol

PCR: The Process Begin with a DNA template Insert in vector 1st strand cDNA Genomic DNA AAAAAAA TTTTTTT

PCR: The Process Denature template Anneal primers AAAAAAA TTTTTTT

PCR: The Process Extend primers with thermostable DNA polymerase Taq Pfu This ends a PCR cycle Additional cycles will repeat these three steps AAAAAAA TTTTTTT

PCR: The Process Beginning of 2nd cycle Melt newly synthesized DNA from template New strands of DNA are now also available as templates Anneal primers Extend primers 1 & 2 3

PCR: The Process Beginning of 3rd cycle Melt newly synthesized DNA from template All new strands of DNA are now also available as templates Anneal primers Extend primers

PCR: Yields How much amplification can be achieved? Each cycle of PCR theoretically doubles the number of template molecules Therefore the rate of amplification is 2n Where n is the number of amplification cycles This will reach a practical maximum yield due to reagent (primer & dNTPs) concentration limits and maximum rate due to limiting enzyme concentrations. This upper limit is about 1x106 X amplification.

PCR: Yields Example: Starting with 2ng of 5kb DNA template to amplify a 1Kb insert, what is the theoretical yield after 20 cycles? After 30? How many template molecules are there? = 5000bp X 660g bp/mol bp = 3.3x106 g template/mol template = 2x10-9 g template  3.3x106 g temp/mol temp = 6x10-16 mol temp = 6x10-16 mol temp X 6.02x1023 molec/mol = 3.64x108 molecules 2. How many molecules of insert can be made in 20 cycles? 30? 3.64x108 molecules x 220 = 3.8x1014 molecules – 106 X 3.64x108 molecules x 230 = 3.9x1017 molecules – 109 X

Using PCR to Map a Molecular Marker Mapping determining linkage distance between loci measure % recombination Molecular marker a locus that does not necessarily encode a protein, yet has allelic variants therefore, no affect on phenotype due to allelic variation will ever exist however, this locus is linked to other loci and could be a useful tool in studying the linked loci Allelic variation in the marker locus is called polymorphism base changes - SNPs sequence length – AFLPs, RFLPs

Marker we will map +/Y; b bibS/b bibS ♂ B/+; b bibS/+ bibL ♂ + + + black bib black bib black bib B/+; b bibS/+ bibL ♂ + bib + bib + bib B/B ; + bibL/ + bibL ♀

Recombination in the F1 females Parental chromosomes black bib + bib Recombinant chromosomes + bib black bib

Mate with b bibS/b bibS male black bib + black bib P black bib + black bib black bib + R bib black bib black +

Analysis +P bP +F1 +F2 bF2 +F2 bF2 Parental Recombinant 750 500 400 300 200 100 50