Recitation 14 Polymerase Chain Reaction PCR repeated replication in order to amplify a speci c region on DNA in vitro in a test tube Players Deoxynucleotides dNTPs Such as dATP dGTP dCTP dTTp Polymerase Primer DNA template Must be complementary to anking region of the ampli cation Must be on the 5 end Players not here that are needed in DNA replication Ligase in PCR we only replicate the leading strand Helicase unzip our strand by heat Steps 1 Denature the strands at 96 Celsius by breaking the hydrogen bonds G C rich DNA requires a higher temperature because there are more hydrogen bonds 2 Annealing sticking on primers at 58 Celsius allowing the primers to hybridize Temperature depends on the length and G C content of primer 3 Elongation to allow DNA polymerase to extend DNA from the 3 end of the primer And then repeat these three steps approximately 35 times How does polymerase work in high heat A special kind of polymerase Taq polymerase is used because it is heat resistant Found in archaebacteria in hot springs DNA sequencing Sanger sequencing 1 Replicate the DNA similarly to PCR 2 Include a few nucleotides that are incorporated but that halt replication They are dideoxy so they cannot make phosphodiester bond and thus replication is halted
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