Zhang Jia, Li Kai, Liao Duanfang, Pardinas Jose R, Chen Linling, Zhang Xu
Genomapping, Inc, Nanhua University, Hengyang, Guangzhou, China.
Lab Invest. 2003 Aug;83(8):1147-54. doi: 10.1097/01.lab.0000081589.91390.df.
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.
随着人类基因组计划的完成，单核苷酸多态性（SNPs）已成为生物医学研究中深入研究的焦点。聚合酶介导的引物延伸已应用于多种SNP分析方法中。然而，这些使用无校对功能聚合酶的SNP分析方法因其可靠性低而受到影响。利用新开发的带有限制性酶切位点EcoR-I的短扩增子，我们能够在广泛的退火温度范围内比较有校对功能和无校对功能的聚合酶在引物延伸中的单碱基区分能力。热力学分析表明，有校对功能的聚合酶具有显著的单核苷酸区分能力。使用未修饰的3'-末端等位基因特异性引物时，有校对活性的聚合酶仅产生模板依赖性产物。利用三种3'-末端修饰的等位基因特异性引物在引物延伸中进一步评估了外切酶（+）聚合酶这种强大的单碱基区分能力。与缺乏3'-外切核酸酶活性的聚合酶在引物延伸中较差的保真度相比，本研究提供了令人信服的证据，即使用有校对功能的聚合酶与3'-末端修饰的等位基因特异性引物相结合，可能是开发SNP分析方法的一种强大新策略。
