Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries.
We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols. To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector. To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP). Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones. We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.
Department of Biochemistry, University of Ottawa, Ontario, Canada.
P
P
Wong
Y
Y
Myal
R
R
Shui
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M
Tenniswood
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015342:DNA Probes; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011388:Prolactin / Q000494:pharmacology; D015183:Restriction Mapping
D015342:DNA Probes; D011388:Prolactin; D004247:DNA; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases