Kasai H, Isono S, Kitakawa M, Mineno J, Akiyama H, Kurnit D M, Berg D E, Isono K
Postgraduate School, Faculty of Science, Kobe University, Japan.
Nucleic Acids Res. 1992 Dec 25;20(24):6509-15. doi: 10.1093/nar/20.24.6509.
We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.
我们开发了一种用于高效分析大肠杆菌K-12基因组序列的策略，该策略通过将Tn5衍生的迷你转座子插入重叠的有序λ噬菌体克隆中以提供通用引物结合位点，并对插入位点附近的DNA片段进行PCR扩增。通过在dnaBamber lacZamber大肠杆菌菌株上形成蓝色噬菌斑来选择含转座子的克隆。每隔0.5 - 1 kb的插入点通过“分析性PCR”确定，转座子插入片段与噬菌体臂之间的片段通过使用一条生物素化引物和一条非生物素化引物的“制备性PCR”进行扩增。扩增的DNA片段单链通过其生物素尾巴与链霉亲和素包被的顺磁珠（Dynabeads M280）偶联，通过磁性纯化，并用作基于荧光的自动核苷酸测序的模板。
