焦磷酸测序测定SNP的常见问题与解决方法

目的:探讨焦磷酸测序技术对单核苷酸多态性分型因测序图谱中存在的一些典型问题而导致分型结果不准确的解决方法。方法:以VKORC1基因1639 GA位点、CYP2C19基因636 GA位点及UGT1A1基因TA重复序列(TA)6(TA)7的多态性检测为例,分别采用优化PCR条件、改变测序时dNTP的加入顺序以及设立外标校正的方法来解决上述问题,从而提高焦测序对SNP分型的准确性。结果:通过升高PCR退火温度,可以显著提高VKORC1基因的扩增特异性,降低了测序图谱中非特异性信号峰强度;通过优化测序时dNTP的加入顺序,CYP2C19基因636 GA位点的准确分型结果可通过观察测序图谱中相关信号峰的有无而简单获得,避免了比较信号峰的相对强度;通过比较待测样本与已知基因型的外标样本的测序图谱来确定待测样本的基因型,提高了对UGT1A1基因TA重复序列(TA)6(TA)7多态性的分型准确性。结论:本文针对焦测序在测定SNP时的常见问题所提出的相应解决方法不仅简单、经济有效,而且在临床应用方面具有可靠性。

Solutions to the Problems Frequently Occurring in Pyrosequencing for Detecting Single Nucleotide Polymorphisms

Objective:To explore the solutions to the problems of inaccurate genotyping results which may be resulted in the typical problems appearing in pyrograms during using pyrosequencing for detecting single nucleotide polymorphisms (SNPs).Methods:Taking the 1639 G>A locus in the VKORC1 gene, the 636 G>A locus in the CYP2C19 gene and the TA repeats (TA)6> (TA)7 in the UGT1A1 gene as examples, the problems were well solved by optimizing PCR conditions, changing the dispensing order of dNTPs and setting up external standards, respectively; the accuracy of SNP genotyping by pyrosequencing was improved significantly.Results:The amplification specificity of the VKORC1 gene was greatly improved by increasing the annealing temperature in PCR, and the intensities of non-specific signal peaks in pyrograms were thus reduced. By optimizing the dispensing order of dNTPs, accurate genotypes of the 636 G>A locus in the CYP2C19 gene were simply achieved by observing the existence of signal peaks instead of comparing the relative intensities of peaks. The genotyping accuracy of the TA repeats (TA)6>(TA)7 polymorphismin the UGT1A1 gene was improved by comparing the pyrogram of a sample with that of each external standard, whose genotype was known in advance.Conclusion:In this article, the solutions proposed to solve the problems frequently occurring in pyrosequencing for detecting SNPs are not only easy and cost-effective, but also reliable in clinical applications.