Evaluation of 15 Polymerases and Phosphorothioate Primer Modification for Detection of UV-induced C:G to T:A Mutations by Allele-specific PCR¶

Abstract

Allele-specific polymerase chain reaction is based on polymerase extension from primers that contain a 3' end base that is complementary to a specific mutation and inhibition of extension with wild-type DNA due to a 3' end mismatch. Taq polymerase is commonly used for this assay, but because of the high rate of nucleotide extension from primer 3' base mismatches documented for Taq polymerase, high sensitivity is difficult to achieve. To determine whether other polymerases might improve assay sensitivity, 15 polymerases were tested with mutation-specific primers for two ultraviolet-induced mutations in the human 5S ribosomal RNA genes. Of the 15 polymerases tested, six were capable of discriminating these mutations at levels equivalent to or better than Taq polymerase. All primers were phosphorothioate modified on the 3' end to block removal of the critical 3' mutation-specific base by polymerases containing 3' --> 5' exonuclease "proofreading" activity. The effectiveness of phosphorothioate modification was measured in mock polymerase chain reaction reactions and a time course. All six enzymes containing this exonuclease activity showed some ability to digest phosphorothioate-modified primers and could be divided into two groups, showing fast and slow digestion kinetics. Of the three enzymes that showed slow digestion kinetics, two also showed significantly slower digestion kinetics of unmodified primers.

Concise Review: Isoforms of OCT4 Contribute to the Confusing Diversity in Stem Cell Biology

“…Nevertheless, it should be cautious when using a primer to distinguish sequences with a single nucleotide variance. Because Taq DNA polymerase, being devoid of 3′ to 5′ exonuclease activity, can make nucleotide extension from the mismatch at the 3′ end of primer by the low reliability [45, 46] (Fig. 3A).…”

“…3A). In addition, polymerases with 3′ to 5′ exonuclease activity, such as Pfx or Pfu DNA polymerase, can proofread the mispairs on the 3′ end of primer by removing the mismatched nucleotide and subsequently make extension along the template [46] (Fig. 3B).…”

Nonspecific PCR amplification by high‐fidelity polymerases: implications for next‐generation sequencing of AFLP markers

“…In designing an AFLP‐sequencing study, the higher error rate of Taq polymerase must be weighed against the lower specificity of proofreading polymerases. Specificity of proofreading polymerases may be improved somewhat by incorporating one or more phosphorothioate bonds at the 3′ end of selective primers (de Noronha & Mullins 1992; Gale & Tafoya 2004). The best approach, however, may be to avoid relying on selective primers for complexity reduction when using high‐fidelity polymerases.…”

Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies1

“…In this review paper, we will first introduce three new assays that we have recently developed, and then describe a number of their applications in pharmacogenetics and in other biomedical studies. nential amplification with phosphorothioate-modified, allelespecific primers [3][4][5][6][7][8][9][10][11][12][13] .…”