Shepard A R, Rae J L
Departments of Physiology/Biophysics and Ophthalmology, Mayo Foundation, 200 1st Street SW, Rochester, MN 55905, USA.
Nucleic Acids Res. 1997 Aug 1;25(15):3183-5. doi: 10.1093/nar/25.15.3183.
We have developed a cDNA library screening method which allows the simultaneous screening of >10 ( 12 ) double-stranded plasmid cDNA molecules with minimal a priori sequence knowledge. A biotinylated, gene-specific oligonucleotide probe along with abutting 'blocking' oligos is hybridized to the plasmid cDNA library and the target plasmid retrieved with paramagnetic streptavidin beads and transformed into Escherichia coli. Multiple rounds of enrichment with a target plasmid represented at 0.002-0.0001% resulted in over one-third positive clones. Our method will be useful for isolating even the rarest cDNAs starting from ESTs, isolated exons or homologous sequence information.
我们开发了一种cDNA文库筛选方法，该方法能够在对先验序列知识要求极低的情况下，同时筛选超过10（12）个双链质粒cDNA分子。将生物素化的基因特异性寡核苷酸探针与相邻的“封闭”寡核苷酸一起与质粒cDNA文库杂交，然后用顺磁性链霉亲和素磁珠回收目标质粒，并转化到大肠杆菌中。对占比0.002 - 0.0001%的目标质粒进行多轮富集，得到了超过三分之一的阳性克隆。我们的方法甚至对于从EST、分离的外显子或同源序列信息开始分离最罕见的cDNA也将是有用的。
