Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping.
Discrimination of haplotype in mitochondrial DNA mixtures using LNA-mediated PCR clamping.
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DOI：
10.1016/j.fsigen.2019.03.018
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发表时间：
2019-07
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通讯作者：
Masaru Asari;Shotaro Isozaki;Chisato Hoshina;K. Okuda;Hiroki Tanaka;Kie Horioka;H. Shiono;K. Shimizu
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文献类型：
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作者：
Masaru Asari;Shotaro Isozaki;Chisato Hoshina;K. Okuda;Hiroki Tanaka;Kie Horioka;H. Shiono;K. Shimizu
锁核酸（LNA）已广泛用于各种遗传分析，并且在扩增的特异性或灵敏度方面具有许多优点，因为与缺乏LNA的探针相比，含有LNA的引物/探针与模板DNA形成更稳定的双链体。在这里，我们开发了一种通过使用 LNA 进行 PCR 钳位来区分线粒体 DNA (mtDNA) 混合物中的 HV1 单倍型的新方法。 PCR 钳位基于使用含有 LNA 的探针选择性抑制扩增，可以区分单核苷酸差异。在设计探针之前，我们从HV1区域选择了171条具有单核苷酸变异的序列，并通过预测Tm值来评估含有LNA的探针对它们的特异性。错配和完全匹配的探针-模板双链体之间的 Tm 差异明显取决于用于区分单核苷酸差异的 LNA 核苷酸类型，并且探针变异位点的胞嘧啶 LNA 核苷酸对于区分这些差异最有效。对于混合物分析，每个探针针对日本人群中特别常见的一种或两种变异（16209C、16217C、16257A/16261T、16297C/16298C、16304C、16362C 或 16362T），并且七种设计的探针完全抑制完全匹配模板的扩增。我们通过将两个个体的 DNA 以 1:9、1:4、1:1、4:1 或 9:1 的比例混合来制备混合样本，然后用每个探针进行 PCR 钳位后进行桑格测序分析。我们的方法以较低的比例从两人混合物中区分出每种单倍型，并能够灵敏检测 12 pg 的总 DNA，包括 600 个 mtDNA 拷贝。此外，我们分析了具有代表性序列的三人混合物，并通过添加两个选定的探针以 10% 的比例检测到一个个体的次要单倍型。通过使用 LNA 介导的 PCR 钳位来区分混合样本中的单倍型的能力表明了 mtDNA 分析在刑事调查中的潜在价值。
Locked nucleic acid (LNA) has been widely used for various genetic analyses, and has many benefits, in terms of the specificity or sensitivity of amplification, because LNA-containing primers/probes form more stable duplexes with template DNA than probes lacking LNA. Here, we developed a new method for discriminating HV1 haplotypes from mitochondrial DNA (mtDNA) mixtures by applying PCR clamping using LNA. PCR clamping is based on the selective inhibition of amplification using LNA-containing probes, which can discriminate single-nucleotide differences. Before designing probes, we selected 171 sequences with single-nucleotide variations from the HV1 region, and evaluated the specificity of LNA-containing probes for them by predicting Tm values. The differences of Tm between mismatched and exactly matched probe–template duplexes depended markedly on the type of LNA nucleotides for discriminating single-nucleotide differences, and the cytosine LNA nucleotide at the site of variations in the probes was most effective to discriminate these differences. For mixture analysis, each probe targeted one or two variations (16209C, 16217C, 16257A/16261T, 16297C/16298C, 16304C, 16362C, or 16362T) that are particularly common in the Japanese population, and seven designed probes completely inhibited the amplification of exactly matched templates. We prepared mixed samples by mixing DNA from two individuals at a ratio of 1:9, 1:4, 1:1, 4:1, or 9:1, and then performed Sanger sequencing analysis after PCR clamping with each probe. Our method distinguished each haplotype at lower ratios from two-person mixtures, and enabled sensitive detection at 12 pg of total DNA including 600 copies of mtDNA. Moreover, we analyzed three-person mixtures with representative sequences, and detected the minor haplotype of one individual present at a rate of 10% by adding two selected probes. The ability to discriminate haplotypes in mixed samples by using LNA-mediated PCR clamping indicates the potential value of mtDNA analysis in criminal investigations.