A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels.
A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels.
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一种新工具，用于从限制性标志基因组扫描凝胶中快速克隆扩增和超甲基化的人类 DNA 序列。
DOI：
10.1006/geno.1999.5840
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Plass,C
中科院分区：
文献类型：
--
作者：
Smiraglia,DJ;Fruhwald,MC;Costello,JF;McCormick,SP;Dai,Z;Peltomaki,P;O'Dorisio,MS;Cavenee,WK;Plass,C
Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments (“spots”) identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.
