摘要
本文发展了ＰＣＲ克隆和亚克隆技术制备ＤＮＡ测序模板。首先，我们用ｐＵＣ／Ｍ１３系列质粒的通用正反向引物ＰＣＲ扩增出质粒ｐＢｌｕｅｓｃｒｉｐｔＫＳＤＮＡ的多克隆位点及其侧翼序列，用ＥｃｏＲＶ和ＸｈｏＩ消化成为左右两个引物多克隆臂，与粘虫核型多角体病毒（ＬｓＮＰＶ）的ＥｃｏＲＶ和ＸｈｏＩ约４００ｂｐ和５００ｂｐ片段分别连接，经ＰＣＲ扩增，得到两端具有上述正反向引物结合位点的测序模板，用ｄｄＮＴＰ链终止法／ＰＣＲ扩增／银染色，从片段两端测定了全部９１９ｂｐ序列，这种ｄｄＮＴＰ／ＰＣＲ／银染测序法简化了操作，大大缩短了测序模板的制备时间，易于实现自动化操作。
Developed a method, termed as PCR clone and sub clone to rapidly preparing sequencing template within a day. First the polylinker and it's flanking regions of pBluescript KS DNA were amplified by PCR with pUC/M13 general reverse and forward primer, then digested with EcoRV and XhoI, resulting in left and right primer arms. The arms were ligated with 400 bp and 500bp fragments of LsNPV DNA/EcoRV XhoI respectively. Enough quantity of foreign DNA with reverse and forward primer binding regions can be amplified using ligation product as a PCR template, and after simply purification, the DNA fragment is available for sequencing. In the paper, the 919bp fragment has been sequenced using ddNTP chain termination/PCR/silver stain sequencing method with reverse and forward sequencing primer. The method developed here simplify the sequencing steps, greatly shortens the time required for preparing sequencing template, and is easily transferred to automation.
出处
《生物工程进展》
CSCD
1998年第4期23-28,共6页
Progress in Biotechnology
基金
国家教委博士点基金
关键词
PCR克隆
银染测序
测序
粘虫
核型多角体病毒
PCR clone Leuscania seperata nuclear polyhedrosis virus Primer polylinker arm Double strand sequencing Silver stain sequencing
