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Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands.
Analytical Methods ( IF 3.1 ) Pub Date : 2020-07-10 , DOI: 10.1039/d0ay00946f
Xinyu Zhuang 1 , Henson L Lee Yu , I-Ming Hsing
Affiliation  

The arrangement of multiple single nucleotide polymorphisms (SNPs) in a gene, called a haplotype phase, is increasingly recognized as critical for accurate determination of disease risk and severity. However, conventional toehold-mediated strand displacement reactions are only able to interrogate SNPs, but not phase them since it is not known whether two SNPs in the same copy of the gene (cis) or in different copies of the same gene (trans) will give the same readout. While the rational introduction of an enzyme enables haplotype phasing, the complicated and stable secondary structure of long, single-stranded DNA sequences at room temperature limits its use. Complex nucleic acid structures make the hybridization of the probes difficult. Thus, we designed a molecular method to reveal the relative positions of SNPs located 1.4 kb apart in two copies of a gene by employing a competitive toehold probes and sink strategy at an elevated temperature. As such, we have successfully differentiated 20 nM of the 10 possible diplotypes in a long DNA target with two SNP sites located 1.4 kb apart within an hour without any additional amplification step. This offers a promising technology for accurate and fast haplotype phasing of SNPs that are over multiple kilobases away from each other.

中文翻译:

基于Toehold探针的询问,用于长核酸链的单倍型定相。

一个基因中的多个单核苷酸多态性(SNP)的排列(称为单倍型阶段)日益被认为对于准确确定疾病风险和严重程度至关重要。但是,传统的脚趾头介导的链置换反应只能审问SNP,而不能对它们进行定相,因为尚不清楚基因的同一拷贝(cis)或同一基因的不同拷贝(trans)将给出相同的读数。虽然合理引入酶可以实现单倍型定相,但室温下长且单链DNA序列的复杂且稳定的二级结构限制了其使用。复杂的核酸结构使探针的杂交变得困难。因此,我们设计了一种分子方法,通过使用竞争性的脚趾探针和在升高的温度下进行沉陷策略,揭示了两个基因拷贝中相距1.4 kb的SNP的相对位置。因此,我们已经成功地在一个长的DNA靶标中,在一个小时内将两个SNP位点相距1.4 kb的10个可能的双型中,将20 nM进行了区分,而无需任何其他扩增步骤。这提供了一种有希望的技术,可以对彼此相距超过数千个碱基的SNP进行准确,快速的单倍型定相。
更新日期:2020-09-03
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