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首页> 外文期刊>Human mutation >Masking selected sequence variation by incorporating mismatches into melting analysis probes.
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Masking selected sequence variation by incorporating mismatches into melting analysis probes.

机译：通过将错配并入解链分析探针来掩盖所选的序列变异。

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摘要

Hybridization probe melting analysis can be complicated by the presence of sequence variation (benign polymorphisms or other mutations) near the targeted mutation. We investigated the use of "masking" probes to differentiate alleles with similar probe melting temperatures. Selected sequence variation was masked by incorporating mismatches (deletion, unmatched nucleotide, or universal base) into hybridization probes at the polymorphic location. Such masking probes create a probe/target mismatch with all possible alleles at the selected polymorphic location. Any allele with additional variation at another site is identified by a lower probe melting temperature than alleles that vary only at the masked position. This "masking technique" was applied to RET protooncogene and HPA6 mutation detection using unlabeled hybridization probes, a saturating dsDNA dye, and high-resolution melting analysis. Masking probes clearly distinguished all targeted mutations from polymorphisms when at least 1 base pair (bp) separated the mutation from the masked variation. We were able to mask polymorphisms immediately adjacent to mutations, except in certain cases, such as those involving single-base deletion probes when both adjacent positions had the same polymorphic nucleotides. The masking probes can also localize mutations to specific codons or nucleotide positions. Masking probes can simplify melting analysis of complex regions and eliminate the need for sequencing.
机译：杂交探针解链分析可能会因目标突变附近存在序列变异（良性多态性或其他突变）而变得复杂。我们研究了使用“掩蔽”探针区分具有相似探针解链温度的等位基因。通过将错配（缺失，核苷酸不匹配或通用碱基）掺入多态性位置的杂交探针中，掩盖了选定的序列变异。这样的掩蔽探针在选定的多态性位置上与所有可能的等位基因产生探针/靶错配。与另一个仅在掩蔽位置发生变化的等位基因相比，在另一个位点具有其他变化的等位基因可以通过更低的探针解链温度来识别。使用未标记的杂交探针，饱和dsDNA染料和高分辨率熔解分析，将该“掩蔽技术”应用于RET原癌基因和HPA6突变检测。当至少1个碱基对（bp）将突变与被掩盖的变异分开时，掩盖探针可清楚地区分所有目标突变与多态性。我们能够掩盖与突变紧邻的多态性，除非在某些情况下，例如当两个相邻位置具有相同的多态性核苷酸时涉及单碱基缺失探针的多态性。掩蔽探针还可将突变定位于特定密码子或核苷酸位置。掩蔽探针可以简化复杂区域的解链分析，并且无需测序。

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