反向嵌套PCR技术扩增驯鹿Ghrelin cDNA 5′末端序列

Rapid Amplification of the 5′cDNA End of Reindeer Ghrelin by Inverse Nested PCR

为了研究Ghrelin在驯鹿生长发育过程中的作用和功能,以驯鹿为研究对象,采用反向嵌套PCR RACE技术原理,根据已知的序列设计1条5′末端磷酸化的特异性反转录引物和2对特异性反向嵌套PCR引物,首先进行反转录(RT),然后将反转录成的cDNA进行环化,最后进行反向嵌套巢式PCR,成功地扩增了驯鹿Ghrelin cDNA 5′末端序列。与锚定PCR相比,反向嵌套PCR法具有特异性强、扩增效率高等优点,是一种非常有效的扩增cDNA 5′末端序列的方法。

In order to study the role and functions in the process of growth and development of the reindeer ghrelin, inverse nested PCR was used for amplifying 5′cDNA end with a specific RT primer which 5′ end is phosphated and two pairs of specific inverse nested PCR primers designed basing on the known cDNA sequence of reindeer ghrelin. The ghrelin cDNA which reversed from ghrelin mRNA is encircled, then inverse nested PCR is performed. Comparing with the anchored PCR , inverse nested PCR has better specificity and higher amplificaton effect, which is a very effective method to amplify 5′cDNA ends.