利用单线虫两重PCR技术对秀丽隐杆线虫线粒体DNA拷贝数进行相对定量的方法

Relative quantification of mitochondrial DNA copy number of Caenorhabditis elegans using single nematode two-plex PCR

目的基于单线虫两重PCR技术,利用细胞核DNA(nDNA)作为内参研究秀丽隐杆线虫线粒体相对数量的实验条件,进而建立一种相对定量秀丽隐杆线虫线粒体DNA(mtDNA)拷贝数的方法。方法首先以秀丽隐杆线虫nDNA和mtDNA序列为模板,利用多重PCR引物设计软件MPprimer和引物特异性评估软件MFEprimer V2.0设计两重PCR引物;然后针对以单线虫为PCR扩增模板的处理条件进行摸索,包括蛋白酶K的消化浓度及反应时间;最后研究秀丽隐杆线虫nDNA片段和mtDNA片段扩增后到达平台期的时间,进而比较不同循环数扩增下秀丽隐杆线虫线粒体拷贝数相对定量的可行性。结果单线虫经蛋白酶K 60℃消化及95℃灭活处理后,能较特异地扩增出nDNA和mtDNA;单线虫PCR模板的处理条件为50μg/ml蛋白酶K 60℃处理2 min并于95℃灭活10 min;线粒体拷贝数相对nDNA而言,趋于稳定的PCR循环数为26。结论建立了一种以为nDNA内参快速相对定量线粒体拷贝数的方法,为线粒体DNA拷贝数相关的实验研究提供了技术支撑。

Objective To optimize the experimental condition for relative quantification of the Caenorhabditis ~elegans mitochondrial DNA（mtDNA） copy number based on single nematode two-plex PCR approach with the cell nuclear DNA （nDNA） as an internal control. Methods The two-plex PCR primers were designed by MPprimer and MFEprimer-v2.0 software according to the mtDNA and nDNA sequences of C. elegans. Subsequently, the experimental conditions of single nematode PCR were investigated,involving the determination of the concentration of proteinase K and the processing time. Finally, the PCR plateau phase of mtDNA and nDNA templates was studied based on the above optimum conditions before the feasibility of C. elegans relative quantification under different amplification cycles was compared. Results mtDNA and nDNA templates could be specifically amplified when the single animal was digested by protease K at 60℃ and deactivated at 95℃, respectively. The experimental conditions for the single nematode PCR included 50 g/ml protease K incubation for 2 min at 60℃ followed by 10 min denaturation at 95℃. The cycle number that tended to be stable .was 26. Conclusion A quick and accurate method has been established for the relative quantification of the C. elegans mtDNA copy number based on the single nematode two-plex PCR approach .with the nDNA as an internal control,contributing much to studies related to mtDNA copy number.