Polymerase Chain Reaction

Polymerase Chain Reaction PCR

PCR Apparatus

Introduction • PURPOSE: to amplify a specific sequence • WHY: sometimes a DNA sample is so small that if you run it on a gel, you won’t see it – requires that you have MANY copies of the sequence

PCR Amplification Process

PROCEDURE • DNA is isolated • Heat at 92ºC – breaks H-bonds, DNA is ss • Heat at 60ºC – add primers anneal (2 diff) • Heat at 70ºC – Taq polymerase elongates 5' to 3' • Repeat cycle about 30X • Cycle 1  variable length strands • Cycle 2+  constant-length strands of target DNA • 30 cycles  230 = 1B • (Treat with endonucleases or probes) • Run on a gel * The machine is referred to as a thermocycler * The technique is PCR

PCR Procedure

How is this useful? • Once the DNA has been amplified, the quantity is large enough to be seen when run on a gel • Bands run differently on gels because of the difference in the # of bps • This can be due to VNTR, LINES, SINES etc

Application • Heredity • Amplify DNA from different family members to check heredity of a specific sequence • Forensics and Paternity/Maternity Testing • Amplify sample of DNA from crime scene to compare to suspects • Presence of diseases • Certain conditions can be diagnosed by the presence of a specific allelic seq or mutation • Quantifying gene expression • PCR with RNA – req sufficient primers for accurate results • Phylogeny (comparing species) • Amplify DNA from a fossil and compare for seq similarity

Polymerase Chain Reaction