聚合酶链式反应偶联单链构象多态性分析(PCR-SSCP)技术用于鹿鞭线粒体DNA指纹的研究

Fingerprint Study of Penis et Testis Cervi mtDNA with PCR-SSCP

目的建立一种快速、简便、准确的分子生物学方法,用来鉴别鹿鞭真伪及品种。方法应用柱色谱法提取梅花鹿鞭、马鹿鞭、牛鞭以及市售鹿鞭线粒体DNA;根据GenBank中鹿类动物线粒体细胞色素b基因序列,设计一对引物用于线粒体DNA的聚合酶链式反应(PCR)特异性扩增;应用变性聚丙烯酰胺凝胶电泳对聚合酶链式反应产物进行单链构象多态分析(single strand conformation polymorphism,SSCP)。结果梅花鹿鞭、马鹿鞭以及部分市售鹿鞭可显示清晰且迁移率不同的片段;对照组伪品鹿鞭及部分市售鹿鞭无特异性显示。结论柱色谱法提取线粒体DNA所需时间短,DNA纯度高,DNA分子的破坏性较小,为特异性聚合酶链式反应扩增提供一级结构保证;单链构象多态分析法可以清晰显示正品梅花鹿鞭、马鹿鞭以及部分市售鹿鞭单链片段,其一级结构中的碱基差别为正品梅花鹿鞭、马鹿鞭的不同迁移率提供依据,也为药源市场鹿鞭的鉴别提供一种可以直接进行亚种间鉴别的快速、简便、准确的可行性方法。

OBJECTIVE To establish a method of molecular biology to identify the authenticity and varieties of Penis et Testis Cer vi rapidly, simply and accurately. METHODS The mitochondrial DNAs （mtDNA） of dried Cervus nippon Penis, Cervus elaphus. L Penis, Penis et Testis Bovis and commercial products were extracted and purified with column chromatography. A pair of primers were designed for PCR special amplification of mtDNA according to cytochrome b gene sequence in the cervidae mtDNA GenBank ; the prod ucts of PCR were analyzed for the single strand conformation polymorphism （SSCP） by denaturing polyacrylamide gel electrophoresis （PAGE）. RESULTS The positive control samples and some commercial products showed clear single strand DNA （ssDNA） bands with different mobility, but the negative control sample and some other commercial products showed nothing. CONCLUSION Col umn chromatography method is a good way for getting mtDNA with high purity and less destruction of structure for the follow-up special PCR amplification; SSCP technique not only shows ssDNA bands of mutually complementing clearly, but also reveals the difference of mobility among ssDNAs from all samples directly. For that reason, the technique of PCR-SSCP will be reliable and feasible to indentify the authenticity and varieties of products from cervidae.