Polymerase Chain Reaction

Initial PCR reactions were performed using reverse primer (50 pmol), 20 ng / template 1 template, 12 cycles of 69 ° C. annealing temperature and then forward and reverse primers only. Another PCR reaction. 29 cycles and an equal amount of primer (10 pmol). PCR products were extracted from agarose gel after gel electrophoresis using gel extraction kit (Qiagen) and ligated to gWiz plasmid between SalI and EcoRV restriction sites using T4 ligase (Intron, Korea) (Intron, Korea ). Aldevron)

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Attempts to document the evolution of the polymerase chain reaction will encounter as many myths as science. The precise fact at least repeated in the literature is that the polymerase chain reaction was conceptualized and executed by Cetus's Kary Mullis et al in the early 1980's. This method was first officially announced at the American Human Genetics Conference in October 1985 and the first clinical application of PCR, the analysis of sickle cell anemia, was announced in the same year. In its original form, PCR is cumbersome and labor intensive. However, the advent of certain DNA sequences that can be isolated from their genomic background and expanded almost indefinitely, will not be a tool for graduate students and late Doctor abuse for a long time. This breakthrough technology comes from the separation and purification of thermostable DNA polymerase.

The purpose of this lecture is to introduce the process of polymerase chain reaction (PCR) and its usage. The polymerase chain reaction can generate large amounts of DNA from very small samples (0.1 ml). There are repeat cycles: separation of double stranded DNA strands and synthesis of complementary strands of each chain. Sample DNA, nucleotides, DNA primers and thermostable DNA polymerase were placed in a PCR apparatus. The sample DNA strand was isolated by heating to 95 ° C. The mixture was cooled to 37 ° C. to bind the primers.