Construction and characterization of a human bacterial artificial chromosome library.
We have constructed an arrayed human genomic BAC library with approximately 4x coverage that is represented by 96,000 BAC clones with average insert size of nearly 140 kb. A new BAC vector that allows color-based positive screening to identify transformants with inserts has increased BAC cloning efficiency. The library was gridded onto hybridization filters at high density for efficient identification of BAC clones by colony hybridization. The library was also formulated into characteristic DNA pools to allow for PCR screening of the library for STS content. We have characterized the library mainly by screening with more than 300 different landmarks that include cDNA, STSs, and cosmid clones. We describe methods for using BAC clones and discuss the implications for genome characterization, mapping, and sequencing.
Kim
U J
UJ
Division of Biology, California Institute of Technology, Pasadena, California, 91125, USA.
Birren
B W
BW
Slepak
T
T
Mancino
V
V
Boysen
C
C
Kang
H L
HL
Simon
M I
MI
Shizuya
H
H
D002460:Cell Line; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods; D003360:Cosmids; D004247:DNA / Q000032:analysis; D005819:Genetic Markers; D005821:Genetic Techniques; D015698:Genomic Library; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping
D005819:Genetic Markers; D004247:DNA