Preparation and screening of an arrayed human genomic library generated with the P1 cloning system.
We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert.
Shepherd
N S
NS
Cancer Research Program, DuPont Merck Pharmaceutical Company, Wilmington, DE 19880-0328.
Pfrogner
B D
BD
Coulby
J N
JN
Ackerman
S L
SL
Vaidyanathan
G
G
Sauer
R H
RH
Balkenhol
T C
TC
Sternberg
N
N
D017101:Bacteriophage P1 / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D005069:Evaluation Studies as Topic; D005347:Fibroblasts / Q000166:cytology; D015894:Genome, Human; D015698:Genomic Library; D006238:Haploidy; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012867:Skin / Q000166:cytology
D017931:DNA Primers