用于多个SNP分阶段的条件置换杂交分析,Analytical Chemistry

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用于多个SNP分阶段的条件置换杂交分析
Analytical Chemistry ( IF 6.7 ) Pub Date : 2017-08-25 00:00:00 , DOI: 10.1021/acs.analchem.7b02300
Tsz Wing Fan ₁ , Henson L. Lee Yu ₁ , I-Ming Hsing ₁

Affiliation

人类基因组的两个染色体拷贝是高度多态的，每条链上的等位基因含量可以决定一个人的生物学结果。尽管许多当前的诊断工具能够同时检测多个突变的存在，但是除非使用长距离PCR或测序技术，或者如果模板在扩增前被分隔成单个拷贝，否则大多数无法确定这些突变的阶段。在此，描述了一种酶联杂交测定法，称为条件置换杂交测定法（CDHA），用于同时快速测定SNP变体的存在和阶段。在这种方法中，短的DNA探针首先用于通过两通道荧光测量定量模板上SNP的数量。然后，在探针和模板之间形成的杂合体为随后的酶置换和荧光团标记的链的淬灭建立了正确的条件，只有当两个SNP都存在于同一链上时，才会发生这种情况。荧光信号的下降由此指示两个SNP的相位。作为概念证明，我们在任意序列的四个变体上测试了该测定方法-在相距100 nts的两个位点上有无突变。本文所述的测定法能够在不到1小时的时间内确定样品的单倍型阶段。该方法有望进行直接的，具有成本效益的，基于实验室的测试，以提取更多的遗传信息，以确定和/或预测依赖于SNP分阶段的疾病和特征。

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Conditional Displacement Hybridization Assay for Multiple SNP Phasing

The two chromosomal copies of the human genome are highly polymorphic, and the allelic content on each strand can dictate a person’s biological outcomes. While many of the current diagnostic tools are able to detect the presence of multiple mutations at the same time, most cannot determine the phase of these mutations unless long-range PCR or sequencing techniques are used or if templates are compartmentalized into single copies prior to amplification. Here, an enzyme-coupled hybridization assay, named conditional displacement hybridization assay (CDHA), is described for the concurrent and rapid determination of the presence and phase of SNP variants. In this approach, short DNA probes were utilized to first quantify the amount of SNPs on the templates using a two-channel fluorescence measurement. The hybrids formed between the probes and the templates then set up the right condition for the subsequent enzymatic displacement and quenching of a fluorophore-labeled strand, which happens only if both SNPs are present on the same strand. The drop in the fluorescence signal thereby indicates the phase of the two SNPs. As a proof of concept, we tested the assay on four variants of an arbitrary sequence—with or without mutation on two sites 100 nts apart. The assay described herein was able to determine the haplotype phase of the samples in less than 1 h. This method promises a direct, cost-effective, and laboratory-based test to extract further genetic information to determine and/or predict diseases and traits dependent on SNP phasing.

更新日期：2017-08-25

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