Polymerase chain reaction, generally known as PCR and also referred to as in vitro DNA amplification, is one of the most widely used techniques in biology labs across the world.
Traditional DNA amplification method
Traditional DNA amplification involves the construction of a vector containing the target gene, followed by the transfer of the vectors into cells to amplify the target gene and then screening by probe. The process includes enzyme digestion, connection, transformation, culture, and probe hybridization. Although there are no technical difficulties for the traditional method, the complicated operation and long cycle length make it suboptimal as a way to amplify DNA. PCR is faster, more efficient, and cheaper, and has many advantages over traditional DNA amplification.
Advantage of PCR cloning
PCR was first theorized by Kary Mullis in 1983 and was later invented in 1985. PCR can amplify traces of DNA and generate millions of copies of a particular DNA sequence. Because of its high sensitivity, specificity, and yield, along with its reproducibility, speed, and convenience, PCR is now widely used in microbiology, medical science, agriculture, and many other fields. PCR has greatly simplified the process of molecular cloning, enabling researchers to much more easily analyze and identify genes of interest.
Basic principles of PCR cloning
At a temperature of 95℃, DNA denatures and yields single-stranded DNA molecules. The primer then binds to a complementary part of the DNA template. The temperature is then lowered to the optimum activity temperature, which is usually around 72℃. This activates DNA polymerase, which synthesizes a new DNA strand complementary to the DNA template in the 5’ to 3’ direction. The DNA template, primers, and polymerase are then thermocycled through denaturation, annealing, and extension steps, allowing DNA polymerase to replicate a target region of DNA by millions of times or more.
PCR has become an essential part of biology labs around the world, and is widely applied to gene cloning, genetic recombination, DNA sequence analysis, and gene quantification. PCR Cloning is also used in cancer gene detection and early diagnosis of hereditary diseases.
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