Since humans have two copies of each gene, multiple mutations in different loci may or may not be found on the same strand of DNA (i.e., inherited from one parent). When a person is heterozygous at more than one position, the placement of these mutations, also called the haplotype phase, (i.e., cis for the same strand and trans for different strands) can result in the expression of different amount and type of proteins. In this work, we described an enzyme-free method to phase two single nucleotide polymorphisms (SNPs) using two fluorophore/quencher-labelled probes, where one of which was biotinylated. The fluorescence signal was obtained twice: first, after the addition of the labelled probes and second, after the addition of the magnetic beads. The first signal was shown to be proportional to the total number of SNP A and SNP B present in the target analyte, while the second signal showed a marked decrease of the fluorescence signal from the non-biotinylated probe when the SNPs were in trans, showing that the probe immobilized on the magnetic bead selectively captures targets with SNPs in a cis configuration. We then mimic the nature of the human genome which consists of two haplotype copies of each gene, and showed that 250 nM of the 10 possible pairs of haplotypes could be differentiated using a combination of fluorescence microscopy and fluorescence detection.

中文翻译：

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寡核苷酸杂交与磁分离测定用于多个 SNP 定相


由于人类每个基因有两个拷贝，不同基因座的多个突变可能会也可能不会在同一条 DNA 链上发现（即从父母之一遗传）。当一个人在多个位置上杂合时，这些突变的放置（也称为单倍型阶段）（即同一链的顺式和不同链的反式）可能导致不同数量和类型的蛋白质表达。在这项工作中，我们描述了一种使用两个荧光团/猝灭剂标记探针（其中一个是生物素化的）对两个单核苷酸多态性（SNP）进行定相的无酶方法。获得两次荧光信号：第一次是在添加标记探针后，第二次是在添加磁珠后。第一个信号显示与目标分析物中存在的 SNP A 和 SNP B 总数成正比，而第二个信号显示当 SNP 处于反式时，来自非生物素化探针的荧光信号显着减少，显示固定在磁珠上的探针选择性捕获具有顺式构型的 SNP 的靶标。然后，我们模拟了人类基因组的性质，该基因组由每个基因的两个单倍型副本组成，并表明使用荧光显微镜和荧光检测相结合可以区分 10 对可能的单倍型中的 250 nM。