酶切-熔解曲线分析:一种新的SNP分型方法及其在中药材鉴定中的应用

Restriction endonuclease digest-melting curve analysis:a new SNP genotyping and its application in traditional Chinese medicine authentication

单核苷酸多态性(single nucleotide polymorphisms,SNP)是一种重要的DNA分子标记,在中药真伪鉴定中有广泛应用。为寻找一种简单快速、稳定性好的SNP分型方法,本研究将限制性内切酶引入熔解曲线分析中,建立了一种新的SNP分型方法,并分别以金银花/山银花、白术/苍术叶绿体序列SNP位点为例,检测了位于或不位于内切酶识别序列区的SNP位点分型能力,结果表明在引物5'端加上长链GC夹子(cggcgggagggcgg)进行扩增,然后选择合适的限制性内切酶酶切,金银花或白术正品酶切产物熔解曲线均为双峰,而混淆品山银花或苍术均为单峰,可实现准确分型,且该方法简单快速、稳定性好,被命名为酶切-熔解曲线分析法(restriction endonuclease digest-melting curve analysis)。

Single nucleotide polymorphisms （SNP） is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp （cggcgggagggcgg） to 5′ end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes rnacrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.