KRT14分子诊断中两种去除假基因干扰的扩增方法比较

Comparison of Two Strategies of Exempting Pseudogene in Molecular Diagnosis of KRT14

目的已报道有3种方法可以去除KRT14P对于KRT14分子诊断的干扰,分别为从活检组织中提取RNA法、用识别KRT14P的限制性内切酶酶切基因组DNA法和长距离特异性扩增KRT14法。本文建立特异性扩增法并和内切酶法就能否完全去除假基因干扰做比较。方法据文献报道KRT14P外显子2上有AluI的酶切位点而KRT14外显子2上没有该位点,用AluI消化基因组DNA。部分消化和完全消化的酶切产物作为模板用于下一步KRT14的分子诊断。比对KRT14和KRT14P的区别,设计特殊引物对使其包括3′末端的碱基和KRT14P错配而和KRT14完全匹配,这些引物对在PCR过程中将只扩增KRT14而去除假基因的干扰。结果部分酶切消化的基因组DNA因为不能完全去除KRT14P可能导致假阳性结果。应用本文中的特殊引物对,能够很好地去除KRT14P的干扰。结论我们采用的特异性扩增法去除KRT14P较酶切法经济有效。

Objective Three strategies were reported in avoiding interference of pseudogenes in molecular diagnosis of Keratin 14 （KRT14） including extracting RNA from tissue of biopsy, digesting genomic DNA with endonucleases recognizing pseudogene of KRT14 （KRT14P） and special amplification of KRT14 from genomic DNA. In this study, we established an approach of special amplification and compared it with the strategy of digesting genomic DNA partly and completely. Methods Endonuclease AluI was adopted to digest genomic DNA which cutting exon2 of KRT14P while leaving exon2 of KRT14 entirely. The products which digested completely and partly would be templates in the next processes of PCR. Based on distinguishes of KRT14 and KRT14P, special primer pairs were designed which mismatched with KRT14P containing 3＇end and matched KRT14 exactly, they would amplify KRT14 exactly and exempt pseudogene in the PCR processes. Results Partly digested genomic DNA would not exempt KRT14P completely which would be amplified in the PCR process and lead to false positive results. With the special primer pairs, we can exempt KRT14P exactly. Conclusions The strategy we adopted of special amplification is more economic and effective than another.