摘要
目的:建立一种高效的扩增线粒体DNA高可变区(mtDNA HVR)的方法。方法:本研究选取5例健康成人静脉血,用人血全基因组DNA试剂盒,提取全基因组DNA,设计引物,用复合PCR方式,对线粒体DNA中的高可变区进行扩增。复合扩增的方式为:用6对套叠引物分开进行两次独立的PCR,扩增mtDNA HVR。第一次扩增用3对引物,目标DNA片段基本涵盖整个线粒体DNA的高可变区,扩增后得到互不重叠的3个短片段,分别为113 bp,126 bp和131 bp。第二次复合扩增用其余的3对引物,目标片段基本重叠在第一次扩增所得的目标片段的区域内,扩增得到3个互不重叠的片段为124 bp,133 bp和93 bp。所有扩增产物经过纯化后测序。结果:复合PCR方式获得的mtDNA HVR基因序列完整,5个样本均出现特异性条带,电泳结果条带单一、清晰。结论:复合扩增PCR方法对mtDNA HVR区的扩增效率高,测序结果稳定,结合6对套叠引物,不但保证了序列的完整性,另外,两次独立的PCR也减少了PCR反应过程中错配的发生,此法也适用于保存时间较久的古代线粒体DNA短片段的研究。复合扩增PCR还展示出了潜在的高产量的特点,相对传统PCR显示了其更多的优势。
Objective： To fred an efficient method for human Mitochondrial DNA Hypervariable Region （mtDNA HVR） extraction. Methods： Five healthy people were selected, using the BloodGen Maxi Kit to extract their genomic DNA and multiplex PCR to amplify mtDNA HVR. We developed two multiplex-PCR reactions of non-overlapping short fragments to cover the whole hypervariable region. Multiplex-I allowed to amplify three non-overlapping fragments of 113, 126 and 131 bp, while Multiplex-II allowed to amplify three non-overlapping fi＇agments of 124, 133 and 93 bp that overlap the fragments obtained by Multiplex-I. Then, purified the PCR-products and sequenced. Results： Multiplex PCR got the complete sequence of mtDNA HVR and were efficient, all of the 5 samples showed spe- cific bands, electrophoresis results single and clear. Conclusion： Multiplex PCR is a high throughput method, its high efficiency, sequenc- ing results were stable as well. Conbined with 6 primer pairs, this method can not only ensure the integrity of the sequence, but also reduce the mismatch occurred during the PCR reaction, and applies to longer lasting short segments of ancient mitochondrial DNA research. Compared with traditional PCR, there are more advantages in the multiplex PCR method.
出处
《现代生物医学进展》
CAS
2013年第7期1360-1363,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(31070835)
陕西省社发公关课题基金项目(2011k15-06-04)
