Shareef Mohammed M, Dancea Horatiu C, Gross Jessica L, Myers Tamara T, Griggs Wendy W, Ahmed Mansoor M, Sheldon David G
Weis Center for Research, Geisinger Clinic, Danville, PA 17822, USA.
Anal Biochem. 2008 Nov 1;382(1):75-6. doi: 10.1016/j.ab.2008.07.003. Epub 2008 Jul 15.
Molecular cloning is an important procedure in molecular biology, but this is often a rate-limiting step and can be very time-consuming, possibly due to low ligation efficiency. Here, we describe a simple polymerase chain reaction (PCR)-based strategy to approach 100% selection efficiency. The post-ligation mixture containing the recombinant was subjected to insert-specific primer-mediated PCR amplification using a high-fidelity DNA Pfu polymerase generating a plasmid containing staggered nicks. The PCR mixture was then digested with endonuclease DpnI, which digests the methylated and hemimethylated parental DNA template. The nicked vector was transformed into XL1 blue supercompetent cells where the nicks were repaired, thus amplifying and selecting only the newly amplified recombinant clones.
分子克隆是分子生物学中的一个重要步骤，但这通常是一个限速步骤，而且可能非常耗时，这可能是由于连接效率较低所致。在这里，我们描述了一种基于简单聚合酶链反应（PCR）的策略，以实现100%的筛选效率。含有重组体的连接后混合物使用高保真DNA Pfu聚合酶进行插入特异性引物介导的PCR扩增，产生含有交错切口的质粒。然后用核酸内切酶DpnI消化PCR混合物，该酶可消化甲基化和半甲基化的亲本DNA模板。带切口的载体被转化到XL1蓝色超级感受态细胞中，在那里切口被修复，从而仅扩增和筛选新扩增的重组克隆。
