古DNA实时荧光定量PCR实验中标准品的制备

实时荧光定量PCR技术通过对PCR每一循环扩增产物的实时检测,可对模板的精确拷贝数进行绝对定量,从而用于古DNA实验中提取和扩增条件的比较和优化.本研究采用异硫氰酸胍碱裂解-SiO2吸附的方法,从采自黑龙江省的晚更新世斑鬣狗化石材料中提取得到了斑鬣狗线粒体基因组古DNA.经常规PCR扩增后,将纯化的扩增产物克隆到微生物体内使其大量复制,再用M13通用引物扩增出含少量外源DNA的古DNA目标片段,从而建立了适用于古DNA荧光定量PCR扩增的标准品的制备方法.经检测分析,运用该方法制备的标准品性质稳定,能够准确地指示反应体系中较为精确的古DNA模板拷贝数,从而反映古DNA的提取和扩增效率,用于比较并优化古DNA提取和扩增条件.

The Preparation of Standard Samples for the Real-Time Amplification of Ancient DNAs

Retrieval of ancient DNA from museum specimens, archaeological finds, fossil and sub-fossil remains has become a methodological aspect in biological evolution studies and phylogeny analysis. Because of the extreme limitation of well- preserved ancient materials, it is important to extract and amplify the maximum amount of genetic information from a minimum of material. Along with the various applications in many related fields, the real time PCR technology can be applied to compare and optimize the extraction and amplification system in ancient DNA experiments, by calculating the exact copy number of the template in each reaction. In this study, we introduce a method to prepare the ancient DNA standard sample, by transferring the purified ancient DNA fragment into E.coli and amplifying the recombinant with M13 universal primers. The standard curve and regression analysis results show that the standard sample can be used as monitor of efficiency of extraction and amplification in ancient DNA experiment.