A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels

Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methyl ation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (re petitive elements or DNA amplifications). me present here the first techniq ue capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles, This technique takes advantage of a plasmid-b ased, human genomic DNA, NotI/EcoRV boundary library, The library is arraye d in microtiter plates, When clones from a single plate are pooled and mixe d with genomic DNA, the resultant RLGS gel is a normal profile with a defin ed set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as web as their pooled rows and column s, Thus, Re have mapped individual RLGS spots to exact plate, row, and colu mn addresses in the library and have thereby obtained immediate access to t hese clones. The feasibility of the technique is demonstrated in examples o f cloning methylated DNA fragments identified in human breast tumor and tes ticular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile. (C) 1999 Academic Pres s.