PCR – Polymerase Chain Reaction

1. PCR – Polymerase Chain Reaction A method of amplifying small amounts of DNA using the principles of DNA replication

2. DNA Replication • Normal DNA replication requires the following: • DNA gyrase • DNA helicase • SSBs • DNA polymerase III andI • DNA ligase • RNA primers and primase • Many nucleotides and template DNA

3. Polymerase Chain Reaction • PCR only requires: • Single strand DNA fragment • Polymerase • Primer • Heat provides the mechanism for H bonds to release, replacing gyrase, helicase, SSBs and primase • DNA primers replace RNA primers

4. PCR Machine

5. Taq Polymerase • Taq polymerases comes from a bacteria that lives in themrmal hotsprings and can tolerate the high heat of PCR (9r degrees C) • These bacteria were discovered in 1966, living in Yellowstone hot springs • The heat tolerant bacteria is essential to PCR

6. PCR Process • DNA sample is heated to 95 degrees • DNA denatures, H bonds break • Sample is cooled to 60 degrees • DNA primers anneal to complementary sections • Taq polymerase enzymes build a complementary strand • DNA sample is heated to 95 degrees…

7. …2 hours later… • In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created. • 20 cycles takes about 1 hour • Amplified sample can be used in DNA fingerprinting, sequencing, recombinant DNA, etc…

8. http://www.sumanasinc.com/webcontent/animations/content/pcr.htmlhttp://www.sumanasinc.com/webcontent/animations/content/pcr.html • http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_6.html