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目的为了获得引物3'端存在SNP点突变的插入缺失遗传标记rs10644346所有等位基因片段。方法基于等位基因特异性PCR原理,设计一条共用上游引物,二条3’端倒数第二个位置特异性碱基的下游引物。运用该三条引物检测150个无关个体及10例亲子关系已确定的三联体,同时运用其中二条引物(共同上游引物和其中一条下游引物)扩增9例样本。结果三条引物扩增150个无关个体均有清晰扩增片段;10例三联体案例亲代与子代扩增片段均符合孟德尔遗传定律;二条引物扩增9例样本后发现特定片段丢失。结论本次研究设计的三条引物PCR,证明特异性碱基位置除了通常的3'末端,理想条件下3'端的其他位置(如倒数第二个位置)也可以成为有效选择,该三条引物设计方法为检测引物侧翼存在点突变的遗传标记提供了一种新的参考。
In order to obtain SNP point mutation at the 3 'end of the primer, all the allelic fragments of rs10644346 were inserted. Methods Based on the allele-specific PCR principle, a downstream primer was designed which shared the upstream primer and the second 3 'end of the penultimate position-specific base. The three primers were used to detect 150 unrelated individuals and 10 triplets with defined parentage, and 9 of them were amplified using two of them (common upstream primer and one of the downstream primers). Results All three unrelated individuals amplified by the three primers had a clear amplified fragment. The amplified DNA fragments of 10 triad cases were all consistent with Mendel 's law of inheritance. Nine fragments were amplified by two primers and the specific fragment was lost. Conclusion The three primer PCR designed in this study proved that the specific base position can be an effective alternative to the usual 3 'end under ideal conditions at other positions (such as the penultimate position) at the 3' end. The three primer design methods It provides a new reference for detecting the genetic markers of point mutations in the primer flanks.