China Pharmaceutical University, Nanjing 210009, China.
Anal Chem. 2010 Mar 1;82(5):2074-81. doi: 10.1021/ac902825r.
Although the pyrosequencing method is simple and fast, the step of ssDNA preparation increases the cost, labor, and cross-contamination risk. In this paper, we proposed a method enabling pyrosequencing directly on dsDNA digested by nicking endonucleases (NEases). Recognition sequence of NEases was introduced using artificially mismatched bases in a PCR primer (in the case of genotyping) or a reverse-transcription primer (in the case of gene expression analysis). PCR products were treated to remove excess amounts of primers, nucleotides, and pyrophosphate (PPi) prior to sequencing. After the nicking reaction, pyrosequencing starts at the nicked 3' end, and extension reaction occurs when the added dNTP is complementary to the non-nicked strand. Although the activity of strand displacement by Klenow is limited, approximately 10 bases are accurately sequenced; this length is long enough for genotyping and SRPP-based differential gene expression analysis. It was observed that the signals of two allele-specific bases in a pyrogram from nicked dsDNA are highly quantitative, enabling quantitative determination of allele-specific templates; thus, Down's Syndrome diagnosis as well as differential gene expression analysis was successfully executed. The results indicate that pyrosequencing using nicked dsDNA as templates is a simple, inexpensive, and reliable way in either quantitative genotyping or gene expression analysis.
虽然焦磷酸测序方法简单快速，但 ssDNA 制备步骤增加了成本、劳动力和交叉污染的风险。在本文中，我们提出了一种可直接对缺口内切酶（NEases）消化的 dsDNA 进行焦磷酸测序的方法。在聚合酶链反应（PCR）引物（用于基因分型的情况）或反转录引物（用于基因表达分析的情况）中使用人工错配碱基引入 NEases 的识别序列。在测序之前，对 PCR 产物进行处理以去除过量的引物、核苷酸和焦磷酸（PPi）。缺口反应后，焦磷酸测序从缺口的 3' 端开始，当添加的 dNTP 与非缺口链互补时，延伸反应发生。尽管 Klenow 的链置换活性有限，但可以准确测序大约 10 个碱基；这个长度足以进行基因分型和基于 SRPP 的差异基因表达分析。结果表明，从缺口 dsDNA 焦磷酸图中两个等位基因特异性碱基的信号高度定量，能够定量测定等位基因特异性模板；因此，成功执行了唐氏综合征诊断和差异基因表达分析。结果表明，使用缺口 dsDNA 作为模板的焦磷酸测序是一种简单、廉价且可靠的方法，可用于定量基因分型或基因表达分析。
