摘要：
DNA polymerases without the 3' exonuclease function ($exo^-$ pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, $exo^-$ polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that $exo^+$ polymerases may exhibit higher nucleotide identification ability when compared to $exo^-$ polymerases for an in vitro genetic analysis. With the application of $exo^+$ polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of $exo^+$ polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of $exo^+$ polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.
作者机构：
[Li, K] Genomapping Inc, Tianjin, Peoples R China.;Jinan Univ, Inst Life Sci & Biotechnol, Guangzhou, Peoples R China.;Chugai Pharma USA, San Diego, CA USA.;Nanhua Univ, Inst Pharm & Pharmacol, Hengyang, Peoples R China.
通讯机构：
[Li, K] G;Genomapping Inc, Tianjin, Peoples R China.
关键词：
exo plus polymerase;proofreading;SNP;3 ' terminal-labeled primer;phosphorothioate
摘要：
The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.
摘要：
With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3′-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo+ polymerases was further evaluated in primer extension using three types of 3′ terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3′ exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3′-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.
摘要：
The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.
作者机构：
Nanhua Univ, Inst Pharmacol & Pharm, Inst Biotechnol, Hengyang 421001, Peoples R China.;Charles R Drew Univ Med & Sci, Vasc Biol Lab, Los Angeles, CA 90059 USA.;Jinan Univ, Inst Life Sci & Biotechnol, Guangzhou, Peoples R China.;Genomapping Inc, Tianjin, Peoples R China.;[Zhang, J; Liao, DF; Fang, WY] Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, China
通讯机构：
[Kai Li] I;Institute of Biotechnology, Institute of Pharmacology and Pharmacy, Nanhua University, Hengyang, China<&wdkj&>Vascular Biology Laboratory, Charles R. Drew University of Medicine and Science, Los Angeles
关键词：
mutagenesis;single nucleotide polymorphisms
摘要：
DNA templates harboring specific single nucleotide polymorphism (SNP) sites are largely needed as positive controls in practical SNP analysis and in determination of the reliability of newly developed methods in high-throughput screening assays. Here we report a one-step method to produce SNP templates by amplifying a wild-type sequence with primers having single nucleotide mismatches at or near their 3' ends. A short amplicon harboring an EcoRI site was used to evaluate the feasibility of our strategy. Perfectly matched primers and primers with a single base mismatch occurring from the first base to the sixth base of the EcoRI site were used for primer extension. By using polymerase without a proofreading function, we kept mismatched nucleotides from occurring in extended primer products, as confirmed by EcoRI digestion and sequencing analysis. The strategy of using primers with a single mismatched base and exo- polymerase was shown to be an efficient one-step method for preparing SNP templates, either for application in the development of SNP screening assays or as positive controls in practical SNP assays.
