MIT7_13f08_lab13_Protocol_PCR

MIT7_13f08_lab13_Protocol_PCR - MIT OpenCourseWare...

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MIT OpenCourseWare http://ocw.mit.edu 7.13 Experimental Microbial Genetics Fall 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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7.13 Fall 2008 Page | 1 PCR with the Platinum PCR SuperMix High Fidelity How PCR works: The polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplification of a specific segment of DNA. The applications of PCR are numerous. They include direct cloning from genomic DNA or cDNA, in vitro mutagenesis and engineering of DNA, genetic fingerprinting, and prenatal diagnosis of genetic defects. The theoretical basis of PCR is outlined in Figure 1, below. There are three nucleic acid segments: the segment of double stranded DNA to be amplified and two single stranded oligonucleotide primers flanking it. Additionally, there is a thermostable DNA polymerase, appropriate deoxyribonucleoside triphosphates (dNTPs), a buffer, and salts. The reaction is performed in a thermocycling machine which can be programmed to repeat the cycle of denaturing, annealing and extension many times. The primers are added in vast excess compared to the DNA to be amplified. They hybridize to opposite strands of the DNA (one to the top strand and one to the bottom strand) with their 3’ ends facing each other so that synthesis by DNA polymerase (which goes 5’ to 3’) extends across the segment between them. In the next cycle each one of the new products can hybridize to the primers and act as a template. The amount of product doubles with every subsequent cycle of synthesis so that 30 cycles should result in a 270 million fold amplification of the exact desired fragment of DNA.
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7.13 Fall 2008
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This note was uploaded on 11/11/2011 for the course BIO 7.012 taught by Professor Lander during the Fall '10 term at MIT.

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MIT7_13f08_lab13_Protocol_PCR - MIT OpenCourseWare...

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