subject: The Fundamental Methods Of Pcr Reagents And Enzymes [print this page] The polymerase chain reaction (or PCR) is a technique for amplifying DNA which was invented by Dr. Kary Mullis in the eighties. It involves multiplying a microscopic strand of DNA by the billions thus creating many more samples with which to conduct tests and experiments and its introduction to scientific technology was thought to be such a valuable contribution that Dr. Mullis won a Nobel Prize for its invention.
The PCR process uses a thermal cycler (also called a DNA Amplifier or PCR machine) which repeatedly raises and lowers the temperature of tubes containing the solution. The heating of the DNA strand separates the DNA helix into two separate strands, parts of which are then used by a DNA polymerase (a replicating enzyme) as templates from which to selectively amplify the target DNA according to instructions it receives from the DNA primer - a synthetically created oligonucleotide.
Originally an enzyme called the Klenow fragment from E. Coli was used in PCR but this proved not to be thermostable and as each heat cycle deactivated it, it had to be constantly replaced slowing down the whole process.
Taq DNA polymerase was originally isolated from a bacterium which lives in the extreme heat of the hotsprings in Yellowstone Park proved to be a better choice than the Klenow fragment but Taq DNA polymerase lacks an essential proof-reading quality, which means much of the amplified DNA it helps create is not error proof.
The overall efficiency of Taq DNA polymerase was found to be improved when used in combination with the Pfu polymerase enzyme found in a single celled organism called Pyrococcus furiosus because unlike Taq DNA polymerase, Pfu is more stable under high temperature and it has a 'proof-reading' ability which enables it to correct any errors that are created during the PCR process.
PCR is highly sensitive and it is essential that all reagents, primers or enzymes used are free from any DNA contamination. To ensure the reliability of the PCR, the preparation of pre-PCR reagents should be done in an area which is has no DNA or RNA contamination.
Pre-PCR - when preparing pre-PCR reagents and enzymes the greatest care must be taken to avoid contamination from nucleic acids, nucleases and heavy metal ions. Only the purest quality water should be used, distilled, deionized and sterilized.
For the sake of consistency, it is best to make large batches of reagents and after testing to ensure its effectiveness, separate into single use samples.
Post-PCR - These reagents should only be used for the detection of amplified DNA. All reagents plasticware, disposables and equipment should only be used for this purpose and not ever be used in pre-PCR procedures nor come into contact with any pre-PCR equipment. If possible the pre-PCR laboroatory (also called the clean room) and the post-PCR lab (called the dirty room) should have separate air systems or at the least air filters to prevent aerosol contamination.
A laboratory should run in a straight line from pre to post PCR with no looping or back steps. The potential for contamination of equipment is just too great.
