Polymerase Chain Reaction PCR PCR Apparatus PCR Apparatus
- Slides: 11
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 30 X 1) 2) 3) 4) 5) § § § 6) 7) Cycle 1 variable length strands Cycle 2+ constant-length strands of target DNA 30 cycles 230 = 1 B (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
