| 李思勤 邹秉杰 王建平 刘云龙 宋沁馨 周国华.焦磷酸测序测定SNP的常见问题与解决方法[J].现代生物医学进展英文版,2014,14(12):2219-2223. |
| 焦磷酸测序测定SNP的常见问题与解决方法 |
| Solutions to the Problems Frequently Occurring in Pyrosequencing forDetecting Single Nucleotide Polymorphisms |
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| DOI： |
| 中文关键词: 焦磷酸测序 PCR 单核苷酸多态性 |
| 英文关键词: Pyrosequencing PCR Single nucleotide polymorphism |
| 基金项目:国家自然科学基金项目（21005088）；江苏省科技支撑计划社会发展项目（BE2010604，BE2012744）；
中国博士后科学基金资助项目（2012M512179，2013T60962） |
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| 中文摘要: |
| 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 G>A 位点、CYP2C19 基因636 G>A 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 G>A 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。 |
| 英文摘要: |
| 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. |
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