Construction of rare restriction site (NotI, SacII and ClaI) linking libraries and sequence-tagged site analysis of single-copy clones from a human chromosome-3-specific library.
We have constructed rare restriction-site (NotI, SacII and ClaI) chromosome 3 (Chr 3)-specific linking libraries in a plasmid-based vector by mass transfer of a lambda phage human Chr-3-specific library (LA03NS01-ATCC57717) into pUC18. Total plasmid DNA isolated from the plasmid-based Chr-3-specific library was digested with either ClaI, NotI or SacII. Linear molecules were separated from undigested circles by pulsed-field polyacrylamide-gel electrophoresis. Purified linear molecules were circularized with T4 DNA ligase and transformed into bacteria. The resulting clones were greatly enriched for sequences recognized by the original restriction endonuclease used for digestion (83 to 95%). These sublibraries are composed of 600 (NotI) 1000 (SacII) or 30,000 (ClaI) clones. Thus, this procedure allows for easy isolation of Chr-3-specific DNA clones containing a variety of rare restriction sites. Sequence-tagged site (STS) data are also presented for five site-specifically mapped Chr-3-specific DNA clones. These studies may facilitate the construction of region specific linking libraries for mapping of various disease-specific loci on Chr 3.
Atchison
L
L
Fox Chase Center, Philadelphia, PA 19111.
Comis
R L
RL
Atchison
M L
ML
D000818:Animals; D010582:Bacteriophage lambda; D001483:Base Sequence; D002893:Chromosomes, Human, Pair 3; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D015723:Gene Library; D008040:Genetic Linkage; D005822:Genetic Vectors; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D013379:Substrate Specificity
D017931:DNA Primers; D004262:DNA Restriction Enzymes; C032824:ATCGAT-specific type II deoxyribonucleases; C035337:CCGCGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases