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POLYMERASE CHAIN REACTION (PCR) Bridges 2014 Polymerase Chain Reaction Simple reaction Produces many copies of a specific fragment of DNA Live replication takes much longer Only requires a small amount of DNA (ng) Many types of PCR What goes into a PCR? Mostly water Autoclaved Forward Primer Front end of target DNA sequence Reverse Primer Back MilliQ end of target DNA sequence Template DNA What goes into PCR? PCR Buffer Balances MgCl2 Helps pH levels target specificity and stabilizes DNA dNTPs Nucleotides (A, G, C, T) Polymerase Polymerase used to “build” target sequence PCR is SENSITIVE! Need specific amounts of each reagent Too Highly subject to contamination Use much or too little can RUIN a PCR sterile technique! Needs specific thermocycling program Thermocycler Thermocycler program Denature Anneal Extend Biological Response to Thermocycler Exponential growth of template DNA 16S rRNA Ribosomal Sequence Ribosomes make proteins All bacteria have 16S Has conserved and hypervariable regions Conserved regions- primer binding site Hypervaribale regions are for species identification