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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用于多重 SNP 定相的寡核苷酸杂交与磁性分离分析

由于人类每个基因有两个拷贝，不同基因座中的多个突变可能会或可能不会出现在同一条 DNA 链上（即从父母一方遗传）。当一个人在多个位置是杂合的时，这些突变的位置，也称为单倍型阶段，（即，对于相同链是顺式，对于不同链是反式）会导致不同数量和类型的蛋白质的表达。在这项工作中，我们描述了一种使用两个荧光团/猝灭剂标记的探针对两个单核苷酸多态性 (SNP) 进行定相的无酶方法，其中一个被生物素化。荧光信号获得两次：第一次，在添加标记探针之后，第二次，在添加磁珠之后。第一个信号显示与目标分析物中存在的 SNP A 和 SNP B 的总数成正比，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降低，表明固定在磁珠上的探针选择性地捕获具有顺式构型的 SNP 的目标。然后我们模拟了由每个基因的两个单倍型拷贝组成的人类基因组的性质，并表明可以使用荧光显微镜和荧光检测的组合区分 10 对可能的单倍型中的 250 nM。显示固定在磁珠上的探针选择性地捕获具有顺式构型的 SNP 的目标。然后我们模拟了由每个基因的两个单倍型拷贝组成的人类基因组的性质，并表明可以使用荧光显微镜和荧光检测的组合区分 10 对可能的单倍型中的 250 nM。显示固定在磁珠上的探针选择性地捕获具有顺式构型的 SNP 的目标。然后我们模拟了由每个基因的两个单倍型拷贝组成的人类基因组的性质，并表明可以使用荧光显微镜和荧光检测的组合区分 10 对可能的单倍型中的 250 nM。