中间产物介导拼接扩增法检测高度降解DNA的初步研究

Pilot study on intermediate-mediated splicing amplification for detecting highly degraded DNA

目的高度降解DNA的遗传检验分析,一直是多个学科领域的难题之一。本文报道一种从降解模板制备较大片段DNA的新方法—中间产物拼接扩增法(IMSA)。方法首先,以高度降解DNA为模板,使用短小扩增子PCR技术,分组合成末端相互重叠的小片段初始扩增产物(<165 bp);而后,将小片段初始扩增产物混合物作为次级模板,以80℃退火进行酶促延伸循环,逐步拼接成长度不等的中间产物(208~316 bp),最终生成大片段扩增终产物(422 bp);随后,用最外侧引物,对大片段IMSA终产物进行PCR倍增。最后,对IMSA终产物进行测序,验证其扩增可靠性。使用甲醛固定石蜡包埋组织DNA样品,验证IMSA方法的实用性。结果IMSA终产物与参照DNA扩增产物长度均为422bp,测序序列完全一致,终产物序列完全忠实于来源DNA。使用本文方法,对于陈旧的甲醛固定石蜡包埋组织,成功实现了较大片段终产物(422bp)的扩增分析。结论针对高度降解DNA缺少大片段扩增模板的问题,本文IMSA方法可以直接拼接合成大片段DNA终产物,预期在法医、考古、病理等领域有较好的应用前景。

Objective Genetic analysis of highly degraded DNA has been a challenge in biology.Here we introduce a novel method of intermediate-mediated splicing amplification(IMSA).Methods First,preliminary intermediates(<165 bp)are obtained by mini-amplicon-PCR from the degraded DNA samples.Then,these short products anneal with each other to the complementary sequence at high annealing temperature(80℃)and are elongated by overlap extension,thus forming the longer intermediates(208 bp^316 bp).Finally,the desired products(422 bp)are generated,which can be amplified at low annealing temperature with the outmost primer pairs.The final IMSA products(422 bp)are sequenced to verify its amplification reliability.Formalin-fixed paraffin-embedded tissue(FFPET)DNA samples are used to verify the practicability of IMSA method.Results The results of Sanger sequencing show that the sequences of ISMA products are completely faithful to the original DNA.This IMSA method is suitable for amplification of FFPET-DNA samples.Conclusion These results suggest that IMSA could be an effective method for genetic analysis of highly degraded DNA in many fields,such as forensic medicine,pathology,and archaeology.