Thursday, April 23, 2009

Polymerase Chain Reaction (PCR): Basic Principle

Polymerase chain reaction (PCR) is a primer mediated enzymatic amplification of specifically cloned or genomic DNA sequences. PCR process was invented by Kary Mullis and it has been automated for routine use in laboratories worldwide. The main purpose of the PCR process is to amplify template DNA using thermostable DNA polymerase enzyme which catalyzes the buffered reaction in which an excess of an oligonucleotide primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies of the target sequence.

Scope of PCR are:
  1. Used in molecular biology and genetic disease research to identify new genes.
  2. Viral targets, such as HIV-1 and HCV can also be identified and quantitated by PCR.
  3. Active gene products can be accurately quantitated using RNA-PCR.
  4. In such fields as anthropology and evolution, sequences of degraded ancient DNAs can be tracked after PCR amplification.
  5. With its exquisite sensitivity and high selectivity, PCR has been used for wartime human identification and validated in crime labs for mixed-sample forensic casework.
  6. In the realm of plant and animal breeding, PCR techniques are used to screen for traits and to evaluate living four-cell embryos.
  7. Environmental and food pathogens can be quickly identified and quantitated at high sensitivity in complex matrices with simple sample preparation techniques.

PCR Process

The PCR process requires a repetitive series of the three fundamental steps that defines one PCR cycle:
  1. Double-stranded DNA template denaturation.

    The DNA sequence which is to be amplified by PCR is known as the template. The double-stranded DNA template must be denatured into two complementary single strands of DNA before the reaction can commence. DNA undergoes rapid denaturation at 940 C. Five minutes are allowed for each denaturation step in a 30 cycle PCR, the enzyme will be subjected to 940 C for a total time of 2.5 hours. In practice, 30 seconds to 2 minutes are allowed for the denaturation step, with 1 minute being the optimum choice for most templates.
  2. Annealing of two oligonucleotide primers to the single-stranded template.

    After denaturation, the reaction is quickly cooled, preventing immediate reannealing of long DNA strands. Due to their small size, oligos now rapidly anneal to the single strands of DNA at positions containing specified template sequence. In these positions, they act as primers for Taq polymerase. Formation of the specific primer-template complex is highly temperature dependent, so for annealing to take place, the temperature of the reaction must be lowered to a preset level calculated to maximize primer-template interaction.
  3. Enzymatic extension of the primers to produce copies that can serve as templates in subsequent cycles.

    Also known as polymerization, this is the final step of the PCR cycle in which the temperature of the reaction is adjusted to the optimum for Taq polymerase activity, which is between 750 C and 800 C. During this step, the polymerase enzyme incorporates nucleotides into the nascent DNA strand, producing a complementary copy of the DNA template in the region specified by the annealed primer. The new temperature is invariably above that at which annealing occurs, but does not lead to denaturation of primer-template complex, presumably because the enzyme is already active at the annealing temperature and significantly increases primer length during the annealing step, y\thus raising its denaturation temperature above that of the polymerization step.


Reference: Number 4 and 11 in References