Introduction of PCR
- Polymerase chain reaction (PCR) is one of the significant advances in DNA based technology.
- It is a rapid, sensitive, inexpensive and simple means of producing relatively large number of copies of DNA molecules from minute copies of source DNA (bacterial, viral, plant and animal).
- It does not depend upon the quality of the source DNA.
- It is powerful tool enabling to detect a single genome of an infectious agent in any body fluid with improved accuracy and sensitivity.
- It can detect many infectious agents, which are missed by routine cultures, serological assays, DNA probes and southern blotting hybridization.
- It is a test tube method to amplify a short, well defined sequence of DNA strand.
- It is carried out biochemically in vitro and does not rely on biological cloning methods.
- It permits the synthesis of millions of copies of nucleotide sequence in few hours.
- It can amplify target sequence less than one part in a million of total initial sample.
- The amplified DNA sequence can be analyzed by southern blotting.
Principle of PCR
- PCR uses 2, oligo-nucleotide primers bearing the complementary sequences to the target gene to direct the amplification of target nucleic acid sequences.
- Repeated rounds of various processes like denaturation, primer annealing and primer extension by thermo-stable enzyme DNA polymerase take place.
- Synthetic oligonucleotide primers are annealed to each single stranded template and DNA polymerase elongates the template in 5’- 3’ direction.
- The specificity of the amplification process lies in the choice of primers.
- It enables making millions of copies of specific DNA sequence, which may be a gene, part of gene or simply a stretch of nucleotides with a known DNA sequence.
- A specimen that may contain the sequence of interest is heated to denature the double stranded DNA.
- The specific synthetic oligonucleotide primers bind to unique DNA sequences of interest and heat stable DNA polymerase extends the primers to create a complete and complementary strand of DNA.
- This process is typically repeated 25-40 times within a period of 3-4 hours thereby creating millions of copies of target DNA sequence.
- If the target DNA sequence were not present in the sample, the primers have nothing to bind to and no amplification occurs.
- Thus, PCR offers potentially unmatched sensitivity and specificity.
- The amplified sequence can be detected under UV light by gel electrophoresis after staining with ethidium bromide and or by hybridization to radioactive or non-radioactive probes after transfer onto synthetic membranes.
- PCR leads to exponential increase in target DNA with repeated cycles.
- Each cycle doubles the amount of DNA in the sample.
- So, PCR amplification is actually the exponential increase of product (P) with PCR cycles (n) i.e.
- The PCR product depends on T, number of template copies you start with.
- But, PCR is not a 100% efficient procedure. PCR product does not quite double each cycle, as given by the equation;
P= T (1 + E), where E= PCR efficiency
- Efficiencies are typically 80-90% and shorter PCR products amplify with greater efficiency.
Polymerase chain reaction (PCR) and its principle