GENOMIC RESTRICTION FRAGMENT CLONING WITH SINGLE PRIMER

定向选择克隆技术是一种极其简单的机制 用于克隆特定的基因组或cDNA片段 单次热复制循环中的寡核苷酸引物。单素数 公司希望将这种方法开发为商业应用。 定向选择克隆感兴趣序列的步骤如下 摘要如下： L--制备双链噬菌体DNA； 2-在一个或多个唯一限制条件下切割双链目标DNA 场地； 3-将噬菌体DNA连接到基因组或cDNA限制性切割 产生与限制性噬菌体DNA相容末端的酶； 4-将所得到的文库转化为合适的大肠杆菌菌株； 5-感染辅助噬菌体以单链形式恢复文库 形式； 6-Anneal：一种针对感兴趣序列的特异引物； 7-通过从底物延伸到形成 双链DNA； 8-选择性地降解单链DNA并转化剩余的DNA 将双链DNA导入到大肠杆菌中。 这项提议的目标是： L-确定拉长底漆单链的最佳条件 模板； 2-确定选择所需双工和对照双工的方法 单链模板转化； 3-制定从基因组中克隆特定片段的协议(或 C DNA)限制性内切酶片段。

The Targeted Selected Cloning technique is an extremely simple mechanism for cloning specific genomic or cDNA fragments using only a single oligonucleotide primer in a single thermal replication cycle. Uniprime Corporation wishes to develop this method for commercial applications. The steps for Targeted Selected Cloning of a sequence of interest are summarized below: l - Prepare double-stranded phagemid DNA; 2 - Cut the double-stranded target DNA at one or more unique restriction sites; 3 - Ligate the phagemid DNA to genomic or cDNA cut with restriction enzymes yielding compatible ends to the restricted phagemid DNA; 4 - Transform the resulting library into a suitable strain of E. coli; 5 - Infect with helper phage to recover the library as a single-stranded form; 6 - Anneal a primer specific for a sequence of interest; 7 - Replicate the annealed templates by extending from the primer to form a double-stranded DNA; 8 - Selectively degrade single-stranded DNAs and transform the remaining double-stranded DNA into E. coli. The goals of this proposal are to: l - Determine optimum conditions to elongate primed single-stranded templates; 2 - Determine methods which select for desired duplexes and against transformation by single-stranded templates; 3 - Develop protocols for cloning specific fragments from genomic (or cDNA) restriction fragments.