The commercial availability of thermostable DNA dependent DNA polymerase enzymes has been the major factor in facilitating the success of PCR amplification. Initially, the Klenow fragment of DNA-dependent DNA polymerase I (involved in replication and repair in the bacterium Escherichia coli), was employed in PCR amplification [Saiki et al., 1985; Mullis et al., 1992; Saiki et al., 1986]. The Klenow fragment is actually a hydrolytic product of the native E. coli DNA-dependent DNA polymerase enzyme which lacks a 5′-3′ exonuclease activity (Fig. 2.3). The Klenow fragment polymerase was used in the first PCR protocols developed, but has the huge disadvantage of exhibiting an optimum reaction temperature at 37°C and being heat labile at the temperatures used in PCR thermocycling reactions. This lability meant that originally, fresh enzyme had to be added after each and every PCR cycle, making the PCR procedure time consuming, labour intensive and highly prone to contamination. Further, it was impossible to generate PCR fragments longer than 400 bp and further processing (using for example Southern blotting or dot spot hybridization), was usually required to identify the presence/absence of specific amplification products from the mixture of amplimers produced.

A breakthrough was achieved however in 1976, when Chien et al. [1976] described a 94kD thermostable DNA-dependant DNA polymerase derived from a eubacterium called Thermus aquaticus, whose natural habitat is hot thermal springs (with ambient temperatures of 70–75°C). This thermostable DNA polymerase or “Taq” enzyme was found to possess similar properties to E. coli DNA dependant DNA polymerase I, with strong homology being found at the amino acid level so: the 3′-OH nucleotide addition site, the dNTP/DNA binding sites, and the 5′-3′ exonuclease sites of the two enzymes (Fig. 7.1). Unfortunately however, though this new Taq polymerase did exhibit some endogenous reverse transcriptase activity, it was found that the 3′-5′ proofreading domain (whereby misincorporated nucleotides are removed from double stranded DNA and replaced by the correct complementary nucleotide) was missing. Moreover, despite the obvious thermoresistant qualities of Taq polymerase, it took a further 2 years for the enzyme to be included in a published PCR protocol [Saiki et al., 1986], after which, there was an explosive increase in interest in PCR mediated amplification protocols.

To date, many different types of thermostable DNA dependent DNA polymerases have been discovered in many different thermophilic organisms (Section 7.5), with the majority of these enzymes having their own particular characteristics and being available from commercial suppliers. However, Taq thermostable DNA polymerase (along with its variants) still remains the most widely used polymerase in PCR reactions performed today.

Keywords

Starch Magnesium Phenol Codon DMSO 

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References

  1. Al Soud W, Johnson LJ, Radstrom P. 2000. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J Clin Microbiol 38:345–350.PubMedGoogle Scholar
  2. Al Soud W, Radstrom P. 1998. Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR inhibiting samples. Appl Environ Microbiol 64:3748–3753.PubMedGoogle Scholar
  3. Al Soud W, Radstrom P. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39:485–493.PubMedCrossRefGoogle Scholar
  4. Burgess LC, Hall JO. 1999. UV light irradiation of plastic reaction tubes inhibits PCR. Biotechniques 27(2):252–256.PubMedGoogle Scholar
  5. Chien A, Edgar DB, Trela JM. 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J Bacteriol 127:1550–1557.PubMedGoogle Scholar
  6. Cline J, Braman JC, Hogrefe HH. 1996. PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24:3456–3451.CrossRefGoogle Scholar
  7. Eckert KA, Kunkel TA. 1993. Fidelity of DNA synthesis catalyzed by human DNA polymerase alpha and HIV-1 reverse transcriptase: effect of reaction pH. Nucleic Acids Res 21:5212–5220.PubMedCrossRefGoogle Scholar
  8. Finke J, Fritzen R, Ternes P, Lange W, Dolken G. 1993. An improved strategy and a useful housekeeping gene for RNA analysis from formalin fixed paraffin embedded tissues by PCR. Biotechniques 14:448–453.PubMedGoogle Scholar
  9. Flaman JM, Frebourg T, Moreau V, Charbonnier F, Martin C, Ishioka C, Friend SH, Iggo R. 1994. A rapid PCR fidelity assay. Nucleic Acids Res 22:3259–3260.PubMedCrossRefGoogle Scholar
  10. Hilali F, Saulnier P, Chachaty E, Andremont A. 1997. Decontamination of PCR reagents for detection of low concentrations of 16S rRNA genes. Mol Biotechnol 7:207–216.PubMedCrossRefGoogle Scholar
  11. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. 1992. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Biotechnol 24:17–27.Google Scholar
  12. Pestoni C. Lareu MV, Rodriguez MS, Muniz I, Barros F, Carracedo A. 1995. The use of the STRs HUMTH01, HUMVWA31/A, HUMF13A1, HUMFES/FPS, HUMLPL in forensic application: validation studies and population data for Galicia (Spain). Int J Legal Med 107:283–290.PubMedCrossRefGoogle Scholar
  13. Rungpragayphan S, Nakano H, Yamane T. 2003. PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries. FEBS Lett 540:147–150.PubMedCrossRefGoogle Scholar
  14. Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. 1986. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele specific oligonucleotide probes. Nature 324:163–166.PubMedCrossRefGoogle Scholar
  15. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. 1988. Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–494.PubMedCrossRefGoogle Scholar
  16. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354.PubMedCrossRefGoogle Scholar
  17. Saunders NJ, Jeffries AC, Peden JF, Hood DW, Tettelin H, Pappuoli R, Moxon ER. 2000. Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58. Mol Microbiol 37:207–215.PubMedCrossRefGoogle Scholar
  18. Wilhelm J, Pingoud A, Hahn M. 2001. Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real time PCR with hybridization probes. Biotechniques 30:1052–1056.PubMedGoogle Scholar
  19. Zimmermann K, Mannhalter JW. 1998. Comparable sensitivity and specificity of nested PCR and single-stage PCR using a thermally activated DNA polymerase. Biotechniques 24:222–224.PubMedGoogle Scholar

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