Intro to PCR PCR (polymerase chain reaction) was invented by Kary Mullis in 1983. Mullis as a chemist working on small nucleotide strands for a biotech company envisioned a faster way of doing things. His discovery has impacted nearly every aspect of biotechnology. For this he won the Nobel peace prize in 1993.

PCR is simply a way to rapidly and accurately make copies of DNA. Accuracy is accomplished through use of primers Complimentary strands of DNA to target (template) DNA Forward primers attach to the 3’ side of the sense strand at the beginning of the targeted region Reverse primers attach to the 3’ side of the antisense strand at the end of the targeted region PCR products are called amplicons What is PCR

Stages of PCR There are 3 stages to PCR reactions Denaturation – separation of strands Annealing – applying the primers Extension – making copies

Denaturation Target DNA is heated to 94C Double stranded DNA must be separated before it can be copied. The heat causes the H-bonds that hold the double helix to release Results in ssDNA (single stranded DNA)

Annealing Primers are added to the ssDNA and are cooled to between 50- 60C Optimum temp for annealing but too hot for the double helix to reform Produces a template-primer hybrid

Extension DNA polymerase is added to replicated the ssDNA at the primers Taq Polymerase is commonly used as it optimally replicates at 72C Was discovered in thermophilic bacteria Extension is 5’-3’ (read 3’-5’ and replicated 5’-3’)

Produces amplicons from the 5’ end of one primer to the 5’ end of the other After 5 PCR cycles 25 copies of the original sequence or 32 copies After 15 PCR cycles 212 copies of the original sequence or 8,192 copies After 25 PCR cycles 222 copies of the original sequence or 4,194,304 copies After 25 PCR cycles 232 copies of the original sequence or 4,294,967,296,copies PCR Products