Herring C D, Chevillard C, Johnston S L, Wettstein P J, Riblet R
Medical Biology Institute, La Jolla, California 92037, USA.
Genome Res. 1998 Jun;8(6):673-81. doi: 10.1101/gr.8.6.673.
We have developed a simple PCR strategy, termed vector-hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation. [The nucleotide sequence data reported in this paper are accessible in GenBank under accession nos. B07512-B07598.]
我们开发了一种简单的PCR策略，称为载体六聚体PCR，其独特之处在于能够轻松地从YAC和BAC载体中的大插入片段克隆中回收每个插入片段末端。我们使用这种方法从覆盖小鼠Igh基因座的YAC重叠群中扩增并分离出所有插入片段末端。从pYAC4载体中四个文库的36个YAC克隆中扩增并测序了77个末端。出乎意料的是，这些YAC的插入片段末端中有40%是LINE1重复序列。非重复末端适合用作消化后的YAC的Southern杂交探针，以鉴定重叠区域并构建重叠群。同样的策略成功地用于从怀特黑德研究所/麻省理工学院-820小鼠YAC文库的pRML载体中的YAC以及pBeloBAC11中的BAC中扩增插入片段末端。这项技术的简单性及其从大插入片段克隆中分离每个末端的能力在基因组研究中具有很大的实用性。[本文报道的核苷酸序列数据可在GenBank中获取，登录号为B07512 - B07598。]
