PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA.
Thank you for reading this post, don't forget to subscribe!PCR is used to reproduce (amplify) selected sections of DNA or RNA. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria, and took weeks. But now, with PCR done in test tubes, it takes only a few hours. PCR is highly efficient in that untold numbers of copies can be made of DNA. Moreover, PCR uses the same molecules that nature uses for copying DNA:
- Two “primers”, short single-stranded DNA sequences that are synthesized to correspond to the beginning and ending of the DNA stretch to be copied;
- An enzyme called polymerase that moves along the segment of DNA, reading its code and assembling a copy; and
- A pile of DNA building blocks that the polymerase needs to make that copy.
How is PCR (polymerase chain reaction) done?
As illustrated in the animated picture of PCR, three major steps are involved in a PCR. These three
steps are repeated for 30 or 40 cycles. The cycles are done on an automated cycler, a device which rapidly heats and cools the test tubes containing the reaction mixture. Each step — denaturation (alteration of structure), annealing (joining), and extension — takes place at a different temperature:
- Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.
- Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded “template” (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.
- Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.
With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template increases exponentially.
Purpose of doing a PCR (polymerase chain reaction)
PCR has become an essential tool for biologists, DNA forensics labs, and many other laboratories that study genetic material.
To do PCR, the original DNA that one wishes to copy need not be pure or abundant. It can be pure but it also can be a minute part of a mixture of materials. So, PCR has found widespread and innumerable uses — to diagnose genetic diseases, do DNA fingerprinting, find bacteria and viruses, study human evolution, clone the DNA of a million years old carcass or remains, establish paternity or biological relationships, etc.
What is RT PCR?
RT-PCR (Reverse transcriptase-polymerase chain reaction) is a highly sensitive technique for the detection and quantitation of mRNA (messenger RNA). The technique consists of two parts:
- The synthesis of cDNA (complementary DNA) from RNA by reverse transcription (RT) and
- The amplification of a specific cDNA by the polymerase chain reaction (PCR).
RT-PCR has been used to measure viral load with HIV and may also be used with other RNA viruses such as measles and mumps.
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Medical Author and editor: Dr Qaisar Ahmed MD, DHMS, Isl. Juri.
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