Polymerase Chain Reaction

The basic method for performing PCR was invented by Kary Mullis, who was awarded the Nobel Prize in Chemistry in October 1993 for this achievement, only seven years after he first published his ideas. Mullis's idea was to develop a process by which DNA could be artificially multiplied through repeated cycles of duplication driven by an enzyme called DNA polymerase.
DNA polymerase occurs naturally in living organisms, where it functions to duplicate DNA when cells divide. It works by binding to a single DNA strand and creating the complementary strand. In Mullis's original PCR process, the enzyme was used in vitro (in a controlled environment outside an organism). The double-stranded DNA was separated into two single strands by heating it to 96°C. At this temperature, however, DNA-Polymerase was destroyed so that the enzyme had to be replenished after the heating stage of each cycle. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process.





Later, this original PCR process was improved by the use of DNA-Polymerase taken from thermophilic (heat-loving) bacteria that grow in geysers at a temperature of over 110°C. The DNA-Polymerase taken from these organisms is thermostable (stable at high temperatures) and, when used in PCR, did not break down when the mixture was heated to separate the DNA strands. Since there was no longer a need to add new DNA-Polymerase for each cycle, the process of copying a given DNA strand could be simplified and automated.
One of the first thermostable DNA-Polymerases was obtained from Thermus aquaticus and called Taq. Taq polymerase is widely used in current PCR practice (May 2004). A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3'->5' proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence. Combinations of both Taq and Pfu are available nowadays that provide both high fidelity and accurate amplification of DNA.











The PCR technique was patented by Cetus Corporation , where Mullis worked when he invented the technique. The Taq polymerase enzyme is also covered by patents. There have been several high-profile lawsuits related to the technique, including most famously a lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds them.

The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands--not more than fifty (usually 18-25 bp) nucleotides that exactly match the beginning and end of the DNA fragment to be amplified. They anneal (adhere) to the DNA template at these starting and ending points, where the DNA-Polymerase binds and begins the synthesis of the new DNA strand.
The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer--not to be confused with the melting temperature of the DNA in the first step of the PCR process--is defined as the temperature below which the primer will anneal to the DNA template and above which the primer will dissociate (break apart) from the DNA template. The melting temperature increases with the length of the primer. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the temperature required to melt it. Melting temperatures that are too high, i.e., above 80°C, can also cause problems since the DNA-Polymerase is less active at such temperatures. The optimum length of a primer is generally from twenty to forty nucleotides with a melting temperature between 60°C and 75°C.
Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine. (See genetic code for further details about codons) Use of degenerate primers can greatly reduce the specificity of the PCR amplification. The problem can be partly solved by using touchdown PCR.

Above mentioned considerations makes primer design very accurate process, on which depends product yield:
• GC-content should be between 40-60.
• Calculated Tm for both primers used in reaction should not differ >5°C and Tm of the    amplification product should not differ from primers by >10°C.
• Annealing temperature usually is -5°C the calculated lower Tm. However it should be chosen    empirically for individual conditions.
• Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
• 3' terminus is extremely case sensitive - it must not be complementary to any region of the    other primer used in the reaction and must provide correct base matching to template.