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 MilliQ Forward Primer Front end of target DNA sequence Reverse Primer Back end of target DNA sequence Template DNA
What goes into PCR? PCR Buffer Balances pH levels MgCl 2 Helps 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 much or too little can RUIN a PCR Highly subject to contamination Use 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