POLYMERASE CHAIN REACTION (PCR)
Polymerase Chain (PCR) is a method widely used to rapidly make millions to billions of copies (complete copies or partial copies) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) to a large enough amount to study in detail. PCR was invented in 1983 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to many of the procedures used in genetic testing and research, including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics.
Principle of Polymerase Chain Reaction
PCR makes it possible to obtain, by in vitro replication, multiple copies of a DNA fragment from an extract. Matrix DNA can be genomic DNA as well as complementary DNA obtained by RT-PCR from a messenger RNA extract (poly-A RNA), or even mitochondrial DNA. It is a technique for obtaining large amounts of a specific DNA sequence from a DNA sample. This amplification is based on the replication of a double-stranded DNA template. It is broken down into three phases: a denaturation phase, a hybridization phase with primers, and an elongation phase. The products of each synthesis step serve as a template for the following steps, thus exponential amplification is achieved.
The polymerase chain reaction is carried out in a reaction mixture which comprises the DNA extract (template DNA), Taq polymerase, the primers, and the four deoxyribonucleoside triphosphates (dNTPs) in excess in a buffer solution. The tubes containing the mixture reaction are subjected to repetitive temperature cycles several tens of times in the heating block of a thermal cycler (apparatus which has an enclosure where the sample tubes are deposited and in which the temperature can vary, very quickly and precisely, from 0 to 100°C by Peltier effect).The apparatus allows the programming of the duration and the succession of the cycles of temperature steps. Each cycle includes three periods of a few tens of seconds.
Requirements of PCR
- A PCR reaction contains the target double-stranded DNA, two primers that hybridize to flanking sequences on opposing strands of the target, all four deoxyribonucleoside triphosphates and a DNA polymerase along with buffer, co-factors of enzyme and water.
- Since the reaction periodically becomes heated to high temperature, PCR depends upon using a heat-stable DNA polymerase.
- Many such heat-stable enzymes from thermophilic bacteria (bacteria that live in high temperature surroundings) are now available commercially.
- The first one and the most commonly used is the Taq polymerase from the thermophilic bacterium Thermus aquaticus.
Steps involved in PCR
The individual steps common to most PCR methods are as follows –
- Extension or Elongation
- Final Elongation
- Final Hold
Advantages of PCR
- PCR (polymerase chain reaction) is an extremely simple yet immensely powerful technique.
- It allows enormous amplification of any specific sequence of DNA provided that short sequences either side of it are known.
- Allow faster diagnosis and identification while enhancing sensitivity and maintaining specificity.
Applications of PCR
PCR already has very widespread applications, and new uses are being devised on a regular basis.
- PCR can amplify a single DNA molecule from a complex mixture, largely avoiding the need to use DNA cloning to prepare that molecule. Variants of the technique can similarly amplify a specific single RNA molecule from a complex mixture.
- DNA sequencing has been greatly simplified using PCR, and this application is now common.
- By using suitable primers, it is possible to use PCR to create point mutations, deletions and insertions of target DNA which greatly facilitates the analysis of gene expression and function.
- PCR is exquisitely sensitive and can amplify vanishingly small amounts of DNA. Thus, using appropriate primers, very small amounts of specified bacteria and viruses can be detected in tissues, making PCR invaluable for medical diagnosis.
- PCR is now invaluable for characterizing medically important DNA samples. For example, in screening for human genetic diseases, it is rapidly replacing the use of RFLPs.
- Because of its extreme sensitivity, PCR is now fundamentally important to forensic medicine. It is even possible to use PCR to amplify the DNA from a single human hair or a microscopic drop of blood left at the scene of a crime to allow detailed characterization.