PCR stands for Polymerase Chain Reaction technique. This technique allowed scientists to generate millions of copies of the specific DNA sample. It is a genetic technique that occurs in vitro which allows the amplification of a targeted region of DNA in an exponential manner. Indeed, if the sequence of interest is present in the DNA extract, it is possible to selectively replicate it (amplification) in very large numbers as well.
History of PCR
Polymerase Chain Reaction (PCR) technique was invented by Kary Banks Mullis, the American biochemist at Cetus Corporation of Emeryville, California in 1983 and was patented in 1984. PCR is widely used in the fields of DNA fingerprinting, DNA sequencing, Forensics, and diagnosis of hereditary diseases as well. PCR allows for fast and inexpensive amplification of DNA fragments because it has the ability to generate a large quantity of DNA from a small amount of nucleic acid. PCR can also be referred to as molecular photocopying.
Characteristics and Principle of PCR
PCR 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, such as denaturation phase, hybridization phase, and elongation phase.
- Denaturation : The two strands of DNA are separated by raising the temperature. This process takes place with 94-95°C and this temperature is called as denaturation temperature. The hydrogen bonds cannot be maintained at the temperature higher than 80°C, hence the double-stranded DNA gets denatured into single-stranded DNA.
- Hybridization : After denaturation the second step to be followed is hybridization, it is carried out at a range of temperature between 40-70°C. By decreasing the temperature, it makes the hydrogen to reform, thus forms complementary strands. The primers which are short single-stranded sequences complementary to regions that flank the DNA to be amplified hybridize more easily than long strand matrix DNA.
- Elongation : The reaction is then heated to 72° C, the optimal temperature for DNA polymerase to act. DNA polymerase extends the primers, adding nucleotide onto the primer in a sequential manner, using the target DNA as a template.
- Primer: The PCR uses a pair of custom primers to direct DNA elongation towards each-other at opposite ends of the sequence being amplified. These primers are typically between 18 and 24 bases in length.
In one cycle, a single segment of the double-stranded DNA amplifies into two separate double-stranded DNA. These two double-stranded DNA is used for amplification in the next cycle to produce its copies. In this way, more copies are generated in Polymerase Chain Reaction.
Applications
- Detection of genetic diseases.
- Detection of infectious diseases.
- Reverse transcriptase PCR (RT-PCR) : Amplification of target mRNA into double stranded DNA and if you reverse it, it generates mRNA from double stranded DNA.
- Diagnosis : It is possible to detect deletions, inversions, insertions, and even point mutations, either by direct analysis of PCR products by electrophoresis or by combining PCR with other techniques
- Amplification of fragment length polymorphism(AFLP): It uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments.
- Restriction fragment length polymorphism(RFLP): It is a technique that exploits variations in homologous DNA sequences, known as polymorphisms, in order to distinguish individuals, populations, or species or to pinpoint the locations of genes within a sequence.
Advantages
- Diagnostic : PCR is used as diagnostic tool for diagnosis of genetic as well as infectious diseases.
- Simplicity : PCR is a simple reaction or the technique for amplification of DNA.
- Efficiency : It is relatively a fast and efficient way of detecting the nucleic acid fragment.
- Sensitivity : This technique is sensitive as it can generate the required samples sequences of DNA.
- Versatility: Versatility due to the genetic sequences from various microorganisms can be identified with the same reaction conditions for diagnosis of different pathologies.
- Specificity : It also offers specificity as it amplifies specific sequence of DNA under strict temperature conditions.
Disadvantages
- If the target sequence is too long then it causes difficulty to ensure that the target sequence will be copied entirely and accurately.
- If a primer sequence appears more than once in a DNA molecule then the primer may attach to more than one area which may cause the experiment to copy the wrong sequence completely.