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 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.
Smiraglia
D J
DJ
Department of Medical Microbiology and Immunology, The Ohio State University, Columbus, Ohio 43210, USA. Smiraglia.1@postbox.acs.ohio-state.edu
Frühwald
M C
MC
Costello
J F
JF
McCormick
S P
SP
Dai
Z
Z
Peltomäki
P
P
O'Dorisio
M S
MS
Cavenee
W K
WK
Plass
C
C
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D005784:Gene Amplification; D015894:Genome, Human; D006801:Humans; D008297:Male; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D004273:DNA, Neoplasm; D004247:DNA; D004262:DNA Restriction Enzymes