应用引物延伸预扩增提高微量DNA拷贝数

To increase copy of small DNA sample by primer-extension preamplification

采用引物延伸预扩增方法 ,可普遍提高微量模板DNA的拷贝数 ,便于进行基因分析时克服标本量少、来源困难的制约。采用常规扩增、检测 2 4 8bp的DYZ1片段体系为观察对象 ,其最小模板量需 1.5ng/2 0 μl体系。以 15个碱基随机寡核苷酸为引物 ,对最小模板量进行预扩增 ,再以其产物 1/10为模板 ,特异扩增DYZ1片段。进行相对定量分析 ,判断原模板DNA拷贝数增加的程度。结果 1.5ng男性DNA经随机扩增后 ,此DYZ1片段拷贝数增加了 10倍以上 ,大大地提高了特异DNA片段扩增的模板量。表明经随机引物延伸预扩增后 ,微量标本DNA片段拷贝数获得普遍提高 。

For random increasing templates DNA quantities in small DNA sample, to overcome limitation of templates DNA quantity and source for genetic analysis. The minimum amount of the template DNA of amplifying a 248bp DYZ1 fragment needs 1.5ng male DNA by ordinary PCR in our lab. First, the 1.5ng DNA sequences was amplified by primer-extension preamplification (PEP)using a mixture of 15-base random oligonucleotides. Then, the one-tenth PEP product was used as templates DNA to amplify the DYZ1 fragments. The DYZ1 product was relatively quantified, we can evaluate the degree of increasing copies of the specific DNA segment after PEP. After the small DNA sample was subjected to PEP, the DYZ1 sequences can be copied no less than 10 times. The results indicate that sequences in the small DNA can be random amplified. This method has implications for the analysis of any small DNA sample.