PCR is a laboratory (in-vitro) technique that is employed Pfor amplifying a targeted portion of DNA. This technique is a cell-free method utilized to synthesize several identical copies of a gene or DNA of interest. Thus, it is a very essential tool in a molecular biology laboratory synonymous to a photocopier as a basic requirement in an office. First, we will outline the requirements for a standard PCR reaction – the following will be needed: DNA polymerase enzyme, magnesium chloride (MgCl2), nucleotides, primers, nuclease free water, target DNA template to be amplified and a PCR machine. The PCR reaction mechanism is simple and starts with an initial Denaturation where the double-stranded DNA is heated to separate the strands (1st step) this allows for the primers to attach to the now single stranded DNA (2nd step). After the primers align to the DNA strand, the DNA polymerase enzyme extends the primers (3rd step) which results into two identical copies of the original DNA template. These three steps are repeated over a series of temperatures and times a single complete process of the three combined is known as an amplification cycle. It is important to optimize every step of the cycle to the DNA template and the set of primers being used. The cycles can be repeated as many times as needed, however, on average a standard PCR is run for around 30 to 40 times after which the amplified product is then analyzed. The polymerase chain reaction is used to amplify a targeted DNA of interest for downstream experimental purposes; and it has also found immense use in detection of pathogenic DNA as well as other applications in genetic testing. PCR is a very sensitive technique as tiny volumes of a single reaction are required, as such it is recommended that a master mix is prepared if there are many reactions.
This story is from the September - October 2020 edition of Scientific India.
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This story is from the September - October 2020 edition of Scientific India.
Start your 7-day Magzter GOLD free trial to access thousands of curated premium stories, and 8,500+ magazines and newspapers.
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