Sun Shuhui, Huang Hao, Qi Yingchuan Billy, Qiu Mengsheng, Dai Zhong-Min
Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University , #16 Xuelin Street, Hangzhou , Zhejiang 310036 , China.
Institute of Developmental and Regenerative Biology, College of Life and Environmental Sciences, Hangzhou Normal University, #16 Xuelin Street, Hangzhou, Zhejiang310036, China; Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, Kentucky, KY40292, USA.
Biotechnol Biotechnol Equip. 2015 Jan 2;29(1):105-110. doi: 10.1080/13102818.2014.988094. Epub 2014 Dec 10.
Traditional cut-paste DNA cloning is often limited by the availability of restriction enzyme sites. Here, we described the complementary annealing mediated by exonuclease (CAME), in which the insert or vector fragment is amplified to carry sequences complementary to the other, and both fragments are modified by exonuleases to create directional single-stranded overhangs. The two recessed DNA fragments are joined through complementary strand annealing. The CAME is highly efficient for cloning the DNA of at least 12 kb and single DNA fragment out of a complex DNA sample. Moreover, the application of CAME greatly improved the efficiency of site-directed mutagenesis.
传统的切割粘贴式DNA克隆常常受到限制酶切位点可用性的制约。在此，我们描述了由核酸外切酶介导的互补退火（CAME），其中插入片段或载体片段经扩增后携带与另一片段互补的序列，并且两个片段都通过核酸外切酶进行修饰以产生定向单链突出端。这两个凹陷的DNA片段通过互补链退火连接在一起。CAME对于克隆至少12 kb的DNA以及从复杂DNA样品中克隆单个DNA片段具有很高的效率。此外，CAME的应用极大地提高了定点诱变的效率。
