Abstract
Polymerases with a proofreading function in their internal 3′ to 5′ exonuclease possess high fidelity for DNA replication both in vivo and in vitro. The obstacle facing Exo+ polymerases for single nucleotide polymorphism (SNP) detection could be bypassed by using primer-3′-termini modification. This hypothesis has been well tested using three types of modified allele specific primers with: 3′ labeling, 3′ to 5′ exonuclease resistance, and 3′ dehydroxylation. Accordingly, three new SNP assaying methods have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of Exo+ polymerases. These new mutation detection assays are widely adaptable to a variety of platforms, including multi-well plate and microarray technologies. Application of Exo+ polymerases to genetic analysis, including genotyping that is mostly relevant to pharmacogenetics, high-fidelity gene expression profiling, rare mutation detection and mutation load assay, will help to accelerate the pace of personalized medicine. 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.
Similar content being viewed by others
Article PDF
References
Zhang J, Li K . The 3′ terminal labeled primer extension: a new method of high throughput screening for SNP analysis. Curr Drug Disc 2001; 9: 21–3.
Zhang J, Li K, Deng Z, Liao D, Fang W, Zhang X . Efficient mutagenesis method for producing the template of single nucleotide polymorphisms. Mol Biotechnol 2003; 24: 105–10.
Zhang J, Li K, Liao D, Pardinas JR, Chen L, Zhang X . Different application of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis. Lab Invest 2003; 83: 1147–54.
Li K, Zhang J . New SNP assays from an old concept of proofreading. Curr Drug Disc 2003; 11: 37–9.
Cahill P, Bakis M, Hurley J, Kamath V, Nielsen W, Weymouth D, et al. Exo-proofreading, a versatile SNP scoring technology. Genome Res 2003; 13: 925–31.
King GC, Di Giusto DA, Wlassoff WA, Giesebrecht S, Flening E, Tyrelle GD . Proofreading genotyping assays and electrochemical detection of SNPs. Hum Mutat 2004; 23: 420–5.
Di Giusto DA, King GC . Strong positional preference in the interaction of LNA oligonucleotides with DNA polymerase and proofreading exonuclease activities: implications for genotyping assays. Nucleic Acids Res 2004; 32: e32.
Zhang J, Meng B, Liao D, Zhou L, Zhang X, Chen L, et al. De novo synthesis of PCR templates for the development of SARS diagnostic assay. Mol Biotechnol 2003; 25: 107–12.
Zhang J, Li K . Single base discrimination mediated by proofreading 3′ phosphorothioate-modified primers. Mol Biotechnol 2003; 25: 223–8.
Zhang J, Li K . On/off regulation of 3′ exonuclease excision to DNA polymerization by exo+ polymerase. J Biochem Mol Biol 2003; 36: 525–8.
Zhang J, Chen LL, Guo ZF, Peng CY, Liao DF, Li K . On/off switch mediated by exo+ polymerases: experimental analysis for its physiological and technological implications. J Biochem Mol Biol 2003; 36: 529–32.
Zhang J, Li K, Pardinas JR, Liao DF, Li HJ, Zhang X . SNP discrimination through proofreading and off-switch of Exo+ polymerase. Mol Biotechnol 2004; 27: 75–80.
Gale JM, Tafoya GB . Evaluation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR. Photochem Photobiol 2004; 79: 461–9.
Di Giusto DA, King GC . Ingle base extension with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays. Nucleic Acid Res 2003; 31: 7.
Bi W, Stambrook PJ . Detection of known mutation by proofreading PCR. Nucleic Acids Res 1998; 26: 3073–5.
Zhang J, Li K . New performance from an old member: SNP assay and de novo sequencing mediated by Exo+ DNA polymerase. J Biochem Mol Biol 2004; 37: 269–74.
Li K, Chen LL, Peng CY, Wang C, Liao DF, Zhang J . Transgenic human assay and evolutionary role of mtDNA by mitochromic analysis. J Nanhua Univ 2004, 32: 1–3.
Li K, Liao DF, Zhang J, Chen LL, Peng CY, Wang C . Mitochromics: a simplified transgenic human assay. Chin J Arterioscler 2003; 11: 601–2.
Author information
Authors and Affiliations
Corresponding author
Additional information
Project supported in part by the National Major Basic Research Program (973) of China (No G2000056905), the Natural Science Foundation of Hu-nan (No 03JJY4041) and the Key Project of Education Committee of Hu-nan.
Rights and permissions
About this article
Cite this article
Liao, Df., Chen, Ll., Peng, Cy. et al. Exo+ proofreading polymerases mediate genetic analysis and its application in biomedical studies. Acta Pharmacol Sin 26, 302–306 (2005). https://doi.org/10.1111/j.1745-7254.2005.00056.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2005.00056.x
Keywords
This article is cited by
-
A study on flooding scenario simulation of future extreme precipitation in Shanghai
Frontiers of Earth Science (2018)