Polymerase Chain Reaction PCR

PCR Apparatus

PCR Apparatus

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 Forensics and Paternity/Maternity Testing 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