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DNA Amplification for Forensic Analysts

DNA polymerase

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DNA polymerases strongly favor incorporation of proper Watson-Crick base pairs. At each template position, there is one correct and three incorrect dNTPs competing for insertion. Nucleotide mis-insertion frequencies are, on average, between 10-3 and 10-5. The three major factors affecting insertion error rates are:

  • The type of mispair formed; G(primer)T and T(primer)G transition mispairs can generally be made more easily than CC or GG transversion mispairs.05
  • The sequence context where the mispair occurs
  • The identity of the polymerase

The PCR process was originally performed manually. The thermolabile Klenow DNA polymerase was used and had to be replenished at the beginning of each cycle. The subsequent introduction of Thermus aquaticus (Taq) polymerase, a thermostable DNA polymerase, represented a considerable advance. Taq DNA polymerase is the most widely used polymerase in forensic DNA analysis and is available from multiple vendors.

Taq DNA polymerase lacks endonuclease and 3' - 5' exonuclease (proofreading) activities, but has 5' - 3' exonuclease activity. Ampli Taq Gold® DNA Polymerase (supplied by Applied Biosystems) is a chemically modified form of Taq DNA Polymerase, which is delivered in an inactive state. A pre-PCR heating step is used to activate the enzyme. Loss of specificity and sensitivity can be caused by competing side reactions that usually occur during the pre-PCR setup period. At this time, all reactants are present in the reaction tube at permissive temperatures, which can result in the amplification of non-target sequences in background DNA either due to mispriming or to primer oligometization.

Enzyme requirements may vary with respect to individual target templates or primers. The recommended concentration range for Taq DNA polymerase is between 1 and 2.5 units per 100µl reaction. However, when conducting studies to optimize a PCR assay, 0.5 to 5 units/100µl is an appropriate range. If the enzyme concentration is too high, nonspecific background products may accumulate; if too low, an insufficient amount of desired product is made.11

Protocols can be enhanced with the addition of proteins (gelatin or bovine serum albumin) and/or nonionic detergents (Tween 20 or Laureth 12). These chemicals are included to help stabilize the enzyme.11

Taq DNA polymerase has the ability to add a single nucleotide to the blunt end DNA fragment, creating a single nucleotide overhang at the 3' end of a product. Although any of the four nucleotides can be added, dATP is preferred. This non-template directed dATP addition occurs during the extension step of the PCR process. The extension step of the process is designed to drive the addition of dATP to ensure that all amplicons are the same length.11

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