Osahor Andrew N, Tan Chau-Yan, Sim Edmund Ui-Hang, Lee Choon-Weng, Narayanan Kumaran
School of Science, Monash University Malaysia, 46150 Bandar Sunway, Selangor, Malaysia; Department of Biotechnology, Malaysia University of Science and Technology, 47301 Petaling Jaya, Selangor Darul Ehsan, Malaysia.
School of Science, Monash University Malaysia, 46150 Bandar Sunway, Selangor, Malaysia.
Anal Biochem. 2014 Oct 1;462:26-8. doi: 10.1016/j.ab.2014.05.030. Epub 2014 Jun 11.
When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
在重组细菌人工染色体（BAC）时，通常的做法是在供体分子的末端设计50个碱基对的同源序列，以指定其插入位点。我们证明，使用短至15个碱基对的分子间同源序列也可以产生所需的重组体。虽然使用较短的供体末端区域会使总重组体数量减少几倍，但具有正确插入供体分子的重组体频率足够高，便于通过简单的聚合酶链反应（PCR）筛选进行检测。这一观察结果可能对重组工程中寡核苷酸的设计具有重要意义，包括显著节省成本，特别是对于使用大量引物的高通量项目。
