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Biology Chapter 19.docx

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Lovaye Kajiura

Biology Chapter 19: Analyzing and Engineering Genes Bernard Ho December 3, 2010 The Polymerase Chain Reaction − The polymerase chain reaction (PCR) is an in vitro DNA synthesis reaction in which a specific section of DNA is replicated over and over to amplify the number of copies of that sequence − It’s a technique for generating many identical copies of a particular section of DNA − Although PCR is much faster and technologically easier than cloning genes into a DNA library, it is only possible when a researcher has some information about DNA sequences near the gene in question − Sequence information is required because to do a polymerase chain reaction, you have to start with short lengths of a single-stranded DNA that match sequences on either side of the gene of interest − These short segments act as primers for the synthesis reaction − Primer sequences must be complementary to bases on either side of the gene in question − One primer is complementary to a sequence on one strand upstream of the target DNA, the sequence of interest − The other primer is complementary to a sequence on the other strand, downstream of the target DNA − If the target DNA molecule is made single stranded, then the primers will bond or anneal to their complementary sequences − Once the primers are bound, DNA polymerase can extend each strand in the 5’ to 3’ direction − A PCR procedure begins with a reaction mix containing an abundant supply of dNTP of the template DNA, copies of the two primers and an enzyme called Taq polymerase o Taq polymerase is a DNA polymerase found in the bacterium Thermus aquaticus, which was originally discovered in a hot spring o Taq polymerase is the enzyme of choice in PCR because the technique depends on heating the reaction mix and because Taq polymerase is heat stable o o Taq polymerase continues to function normally even when heated to 95 C o − PCR gets under way when the reaction mix is heated to 94 C o At this temperature, the double-stranded template DNA denatures o This means that the two DNA strands separate, forming single-stranded templates o o Then, the mixture is allowed to cool to 50-60 C o In this temperature range, some of the denatured DNA strands re-form double helices − But some of the primers bond or anneal to complementary portions of the single- stranded template DNA o This step is called primer annealing o − The reaction mix is then heated to 72 C o At this temperature, Taq polymerase synthesizes the complementary DNA strand from the dNTPs, starting at the primer o This step is called extension o The temperature changes required in each step are now automated by PCR machines − The denaturation, primer annea
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