Cloning of no tI-cleaved genomic DNA fragments appearing as spots in 2D gel electrophoresis.
RLGS (Restriction Landmark Genomic Scanning) is a simple and rapid scanning of genomic DNA in two-dimensional electrophoresis. Human genomic DNA is first cleaved by NotI, and the cleaved ends are radio-labeled and cleaved further by EcoRV, followed by size-fractionation by first dimensional electrophoresis. The sample is then cleaved in situ by the second enzyme HinfI and resolved by the second dimensional electrophoresis. Nearly 2,000 spots emerge with spot intensities reflecting the copy number in the genome. Because of the resolving power and capacity to scan the entire genome, RLGS has been used to monitor genomic aberrations and imprinting. Here, we report a means of cloning the DNA in spots. The DNA was eluted and ligated to biotinylated NotI and HinfI likers followed by affinity separation using streptavidin. The ligated fragment was recovered by EcoRV cleavage, the target sequence of which was located in the NotI linker and amplified by PCR using a primer pair, the sequences of which lie in the linkers. The products were then cloned into a vector for further tests. An amplified spot in stomach cancer genomic DNA and a dwindling spot in liver cancer genomic DNA were taken as examples for cloning.
Nagai
H
H
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Pongliktmongkol
M
M
Kim
Y S
YS
Yoshikawa
H
H
Matsubara
K
K
D001483:Base Sequence; D001710:Biotin; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015183:Restriction Mapping; D013379:Substrate Specificity
D001710:Biotin; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases