Chapter_8_-_Polymerase_Chain_Reaction_v2

Chapter_8_-_Polymerase_Chain_Reaction_v2 -...

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8. Polymerase Chain Reaction Kary B. Mullis received the Nobel Prize in Chemistry in 1993 for discovery of of PCR Thermostable polymerase called Taq polymerase isolated from Thermophilus aquaticus is a thermophilic archaebacterium that thrives in hot springs near the boiling point Taq polymerase has high processivity (adds successive nucleotides without dissociating from the DNA) Like all DNA polymerase Taq requires a primer Taq lacks proofreading activity, so it is unable to correct erroneously incorporated bases PCR is the exponential amplification of a DNA template To carry out PCR, at minimum, flanking sequence must be known to make PCR primers
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9. Polymerase Chain Reaction 8.1 The PCR Reaction One primer designed to bind to sense strand, other to antisense strand Denaturation at 94 o C to separate the two strands (30 seconds – 1 min) Annealing at 40 o C to 60 o C for hybridization of primers to template (1 min); hybridization temperature depends on GC content and length of primers Extension at 72 o C, where Taq adds nucleotides to the 3’OH at ~ 1kb per minute Programmable thermocycler heats and cools PCR reactions (in 25 or 50 ul volumes, with 0.2 ml thin walled PCR tubes) Hot bonnet lid or mineral oil overlay prevents condensation on the lids of the PCR tubes Figure 8.1 Polymerase chain reaction: first cycle
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8. Polymerase Chain Reaction 8.1 The PCR Reaction Primers will bind to the original template in the second cycle, but also to the strands synthesized in the first cycle Exponential doubling of products the lie between the primers in every cycle After 30 cycles, there is 2 30 > 1 billion copies of the original strand Figure 8.2 Polymerase chain reaction: second cycle
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8. Polymerase Chain Reaction 8.1 The PCR Reaction The ends of the new DNA in the PCR has the primer sequence at either end Modifications such as restriction sites can be introduced at the 5’ end of the primers In the first cycle of the PCR, the 5’ end restriction site sequence of the primer is a flap that does not hybrize to the template (most critical sequence for template
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This note was uploaded on 07/10/2010 for the course BIOL 208 taught by Professor Chuong during the Spring '09 term at Waterloo.

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Chapter_8_-_Polymerase_Chain_Reaction_v2 -...

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