Polymerase Chain Reactions

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Presentation transcript:

Polymerase Chain Reactions Revised May 2010

What is PCR? Technique which takes a small DNA sequence and makes it usable for further testing Faster way of cloning specific DNA Developed in the 1980s by Kary Mullis, who won a Nobel Prize for her research PCR targets segments at the end of each sequence it wants to amplify Number of targets can vary

Primers Primers are strands of DNA 15 to 30 nucleotides long used as the complimentary building blocks of the target sequence Primers for each target sequence is needed for DNA to be copied properly Primers should be added in excess to prevent DNA strands from binding back on each other

Taq Polymerase Produces an enzyme called DNA polymerase which, in the presence of magnesium, amplifies the DNA between the primers Named for the Thermus aquaticus microbe found in Yellowstone National Park

PCR Process Denaturation Annealing Elongation of the strand at 72o Separate DNA strands by heating them to 95o Annealing Allowing DNA to form hydrogen bonds by cooling to 55o Elongation of the strand at 72o There are many cycles in the process, usually between 25 and 40. Takes place in a thermal cooler

Why Use PCR? Diagnose Chlamydia trachomatis and Neisseria gonorrhea, two sexually transmitted diseases Part of the HIV-1 test to check the severity of the disease Test for mutations in Factor V Leiden, which can cause blood clots Forensic testing