RT一PCR法制备NGFcDNA

Preparation of NGF cDNA by RT一PCR in total RNA

本研究直接以新生１８～２１天龄的小鼠脑的全ＲＮＡ（含ＮＧＦｍＲＮＡ）为底物，进行逆转录一聚合酶链反应（ＲＴ一ＰＣＲ），制备出了神经生长因子（ＮＧＦ）的ｃＤＮＡ片段。用ｓａｎｇｅｒ双脱氧链终止法测序分析表明，所制备的ＮＧＦｃＤＮＡ片断为３６３ｂｐ，与编码成熟ＮＧＦ的ｍＲＮＡ碱基顺序相对应。直接利用全ＲＮＡ进行ＲＴ与ＰＣＲ一步联合反应，不仅简化了实验步骤，而且最大程度地减少了样品的需要量，减小了样品可能损失或破坏的机率，提高了反应效率。

We described here a simplified protocol for preparation of NGFcDNA using RT一PCR method.The total RNA from the brain of 18～ 2l days old newborn mice was directly used as sub-strate. The nucleotide sequence and analysis by means of dideoxynucleotide chain-terminator method showed that the prepared c DNA fragment was 363 bp nucleotide and corresponded to the mRNA coding the amino acid sequence of mature NGF.