State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Biomed Res Int. 2017;2017:1397902. doi: 10.1155/2017/1397902. Epub 2017 Dec 10.
We develop a strategy for haplotype analysis of PCR products that contained two adjacent heterozygous loci using sequencing with specific primers, allele-specific primers, and ddNTP-blocked primers. To validate its feasibility, two sets of PCR products, including two adjacent heterozygous SNPs, ⁎6 (rs4148323) and ⁎28 (rs8175347), and two adjacent heterozygous SNPs, K1637K (rs11176013) and S1647T (rs11564148), were analyzed. Haplotypes of PCR products, including ⁎6 and ⁎28, were successfully analyzed by Sanger sequencing with allele-specific primers. Also, haplotypes of PCR products, including K1637K and S1647T, could not be determined by Sanger sequencing with allele-specific primers but were successfully analyzed by pyrosequencing with ddNTP-blocked primers. As a result, this method is able to effectively haplotype two adjacent heterozygous PCR products. It is simple, fast, and irrespective of short read length of pyrosequencing. Overall, we fully hope it will provide a new promising technology to identify haplotypes of conventional PCR products in clinical samples.
我们开发了一种使用特定引物、等位基因特异性引物和 ddNTP 阻断引物对包含两个相邻杂合位点的 PCR 产物进行单倍型分析的策略。为了验证其可行性，我们分析了两组 PCR 产物，包括两个相邻的杂合 SNP ⁎6(rs4148323)和 ⁎28(rs8175347)，以及两个相邻的杂合 SNP K1637K(rs11176013)和 S1647T(rs11564148)。通过使用等位基因特异性引物进行 Sanger 测序，成功地分析了 ⁎6 和 ⁎28 的 PCR 产物的单倍型。此外，虽然使用等位基因特异性引物的 Sanger 测序无法确定 K1637K 和 S1647T 的 PCR 产物的单倍型，但使用 ddNTP 阻断引物的焦磷酸测序却可以成功地进行分析。因此，该方法能够有效地对两个相邻的杂合 PCR 产物进行单倍型分析。它简单、快速，并且不受焦磷酸测序短读长的限制。总的来说，我们完全希望它将为鉴定临床样本中常规 PCR 产物的单倍型提供一种有前途的新技术。
