BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Single-base Discrimination Mediated by Proofreading Inert Allele Specific Primers

  • Lin-Ling, Chen (SNP Institute, Nanhua University) ;
  • Zhang, Jia (Genomapping Inc.) ;
  • Sommer, Steve S. (Molecular Genetics and Human Genomics, City of Hope National Medical Center) ;
  • Li, Kai (SNP Institute, Nanhua University)
  • Published : 2005.01.31
Download PDF
⟨ Previous Next ⟩

Abstract

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by $exo^+$ DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.

Keywords

References

  1. Bi, W. L. and Stambrook, P. J. (1998) Detection of known mutation by proof-reading PCR. Nucleic Acids Res. 26, 3073- 3075 https://doi.org/10.1093/nar/26.12.3073
  2. Cahill, P., Bakis, M., Hurley, J., Kamath, V., Nielsen, W., Weymouth, D., Dupuis, J., Doucette-Stamm, L. and Smith, D. R. (2003) Exo-proofreading, a versatile SNP scoring technology. Genome Res. 13, 925-931 https://doi.org/10.1101/gr.939903
  3. Chicurel, M. (2001) Faster, better, cheaper genotyping. Nature 412, 580-582 https://doi.org/10.1038/35088146
  4. Di Giusto, D. and King, G. C. (2003) Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays. Nucleic Acids Res. 31, 7-18 https://doi.org/10.1093/nar/gng007
  5. Di Giusto, D. A. and King, G. C. (2004) Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays. Nucleic Acids Res. 32, 32- 39 https://doi.org/10.1093/nar/gnh036
  6. Goodman, M. F. and Fygenson, D. K. (1998) DNA polymerase fidelity: from genetics toward biochemical understanding. Genetics 148, 1475-1482
  7. Guo, Z., Zhang, J., Liao, D., Yang, X., He, S. and Li, K. (2003) Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers. Chin. Med. Genet. J. 20, 328-330
  8. Kunkel, T. A., Sabatino, R. D. and Bambara, R. A. (1987) Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc. Natl. Acad. Sci. USA 84, 4865-4869 https://doi.org/10.1073/pnas.84.14.4865
  9. Li, K. and Zhang, J. (2003) New SNP assays from an old concept of proofreading. Current Drug Discovery 11, 37-39
  10. Liu, Q. and Sommer, S. S. (2000) Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification. Biotechniques 29, 1072-1080
  11. Mattila, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991) Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase: an extremely heat stable enzyme with proofreading activity. Nucleic Acids Res. 19, 4967-4973 https://doi.org/10.1093/nar/19.18.4967
  12. Zhang, J. and Li, K. (2001) The 3' terminal labeled primer extension: a new method of high throughput screening for SNP analysis. Curr. Drug Discovery 9, 2124
  13. Zhang, J., Liao, D., Yan, F., Zhang, X. and Li, K. (2001) Development of new generation of biochips for direct genetic analysis. Am. J. Chin. Clin. Med. 3, 312-315
  14. Zhang, J. and Li, K. (2003a) Single base discrimination mediated by proofreading 3 phosphorothioate-modified primers. Mol. Biotechnol. 25, 223-228 https://doi.org/10.1385/MB:25:3:223
  15. Zhang, J. and Li, K. (2003b) On/off regulation of 3' exonuclease excision to DNA polymerization by exo+ polymerase. J. Biochem. Mol. Biol. 36, 525-528 https://doi.org/10.5483/BMBRep.2003.36.6.525
  16. Zhang, J., Li, K., Deng, Z., Liao, D., Fang, W. and Zhang, X. (2003a) Efficient mutagenesis method for producing the templates of single nucleotide polymorphisms. Mol. Biotechnol. 24, 105-110 https://doi.org/10.1385/MB:24:2:105
  17. Zhang, J., Li, K., Liao, D., Pardinas, J. R. and Zhang, X. (2003b) Different applications of polymerases with and without proofreading activity in SNP analysis. Lab. Invest. 83, 1147- 1154 https://doi.org/10.1097/01.LAB.0000081589.91390.DF
  18. Zhang, J., Chen, L., Guo, Z., Peng, Z., Liao, D. and Li, K. (2003c) On/off switch mediated by exo+ polymerases: Experimental analysis for its physiological and technological implications. J. Biochem. Mol. Biol. 36, 529-532 https://doi.org/10.5483/BMBRep.2003.36.6.529
  19. Zhang, J., Meng, B., Liao, D., Zhou, L., Zhang, X., Chen, L., Guo, Z., Peng, C., Zhu, B., Lee, P. P., Xu, X., Zhou, T., Deng, Z., Hu, Y. and Li, K. (2003d) De novo synthesis of PCR templates for the development of SARS diagnostic assay. Mol. Biotechnol. 25, 107-112 https://doi.org/10.1385/MB:25:2:107
  20. Zhang, J., Li, K., Pardinas, J. R., Liao, D. F., Li, H. J. and Zhang, X. (2004) SNP discrimination through proofreading and off-switch of $exo^+$ polymerase. Mol. Biotechnol. 27, 75-80 https://doi.org/10.1385/MB:27:1:75

Cited by

  1. Enhanced discrimination of single nucleotide polymorphism in genotyping by phosphorothioate proofreading allele-specific amplification vol.369, pp.1, 2007, https://doi.org/10.1016/j.ab.2007.04.042
  2. Molecular marker systems in insects: current trends and future avenues vol.15, pp.11, 2006, https://doi.org/10.1111/j.1365-294X.2006.03014.x