Two-dimensional separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments.
The two-dimensional (2-D) separation of genomic digests has provided the means to analyze over 2000 unique restriction fragments simultaneously in a single gel, for genetic variation as well as for genomic alterations in cancer. By utilizing different combinations of restriction enzymes or different electrophoretic conditions, the number of analyzable fragments in multiple 2-D patterns can be augmented. We have previously shown the feasibility of distinguishing between spot intensities representing fragments from one allele and from two alleles and have implemented approaches for the cloning of fragments of interest in 2-D gels. In this study, the 2-D separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments was performed. Three hundred forty-six NotI fragments in whole genomic preparations were assigned to chromosome 1. To verify the reliability of the assignment, two of the NotI fragments attributed to chromosome 1 were cloned and sequenced. The fragments that contained CpG islands were mapped by FISH to 1p35-p36.1 and to 1p13.3-p21, respectively. Our study indicates the feasibility of analyzing 2-D separations of whole genomic digests for the detection of alterations in specific chromosomes. The large number of restriction fragments attributed to chromosome 1 provides the means to screen 2-D patterns for chromosome 1 deletions and amplifications with a high marker density.
Wimmer
K
K
Department of Pediatrics, University of Michigan, School of Medicine, Ann Arbor 48109, USA.
Thoraval
D
D
Asakawa
J
J
Kuick
R
R
Kodaira
M
M
Lamb
B
B
Fawcett
J
J
Glover
T
T
Cram
S
S
Hanash
S
S
D002460:Cell Line; D002878:Chromosomes, Human, Pair 1; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D011110:Polymorphism, Genetic
D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; C038565:GCGGCCGC-specific type II deoxyribonucleases