1. Polymerase Chain Reaction
PCR

2. PCR Apparatus

3. PCR Apparatus

4. PCR Apparatus

5. 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

6. PCR Amplification
Process

7. 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

8. PCR Procedure

10. 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

11. 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