LIGATION-INDEPENDENT CLONING OF PCR PRODUCTS (LIC-PCR)
LIGATION-INDEPENDENT CLONING OF PCR PRODUCTS (LIC-PCR)
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DOI：
10.1093/nar/18.20.6069
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发表时间：
1990-10-25
影响因子：
14.9
通讯作者：
DEJONG, PJ
中科院分区：
文献类型：
--
作者：
ASLANIDIS, C;DEJONG, PJ
已经开发了一种新的程序来有效地克隆复杂的PCR混合物，从而导致库仅由重组克隆组成。重组是在PCR产物和PCR扩增的质粒载体之间产生的。该过程不需要使用限制酶，T4 DNA连接酶或碱性磷酸酶。用于生成可克隆的PCR片段的引物的5'' - 末端包含缺乏DCMP的12个核苷酸（NT）序列。结果，放大产品包括在其3'' - 末端缺乏DGMP的12-nt序列。 3'' - 末端序列可以通过（3'''。fwdarw.5'）的作用去除T4 DNA聚合酶在DGTP存在下的作用，从而导致5''' - 延伸单链的片段（SS）定义序列和长度的尾巴。同样，整个质粒载体被与多个克隆位点同源的引物放大。载体寡核酸具有与用于碎片扩增的尾巴相互互补的12 nt尾巴，从而在DCTP存在下允许用T4 DNA聚合酶创建SS端。矢量分子和PCR片段之间可能发生圆形化，这是由12-nt凝聚末端介导的，但在缺乏插入片段的混合物中不进行。所得的圆形重组分子不需要体外结扎即可进行有效的细菌转化。我们已经将程序应用于杂交细胞线和人类宇宙克隆的alu间片段的克隆。
A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5''-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3''-ends. The 3''-terminal sequence can be removed by the action of the (3''.fwdarw.5'') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5''-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.