摘要
引物是决定 PCR 结果的关键。本实验中,引物Ⅰ、Ⅱ虽有26个碱基,但由于其5’端有8个碱基为限制性内切酶的识别序列。所以实际上只有18个碱基与原始模板互补配对。采用标准的 PCR 方法时不能产生理想结果。经采用 PCR——四步两段扩增法从人胎盘cDNA 文库中分离得到—484bp 的 DNA 片段。通过斑点杂交鉴定,结果表明该 DNA 片段为 hbFGF 基因。
Primers are the key factor to decide the result of PCR.In this experiment,there are twenty-six bases in primer Ⅰ and primer Ⅱ respectively,but only eighteen bases can he paired with original template DNA because eight bases at 5'-end of the primers are restriction en- donuclease cleavage sites.No good result can be obtained by standard PCR method.PCR-Four Steps Two Stages method is developed and hbFGF gene is obtained from human placenta cDNA library by that method,hbFGF gene is identified by dot hybridization under stringent hybridiza- tion and posthybridization washing temperature which can make difference between the hybrid completely pairing and the hybrid one base wrong pairing.The result of dot hybridization shows that the DNA fragment obtained by PCR-Four Steps Two Stages method is exactly the hbFGF gene. In PCR-Four Steps Two Stages method,there are two stages under different conditions.In the first stage,there are four steps which are denature at high temperature,anneal at lower tem- perature,extension at low temperature for a short time and extension at middle temperature.In the second stage,there are three steps of standard PCR which are denature at high temperature, anneal at low temperature and extension at middle temperature.
出处
《生物工程进展》
CAS
CSCD
1997年第1期52-55,共4页
Progress in Biotechnology
关键词
碱性成纤维细胞
生长因子
基因
四步两段扩增法
PCR-Four Steps Two Stages Method
Human basic fibroblast growth factor(hbFGF)gene
Dot hybridization
