Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.
Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.
Zabarovsky
E R
ER
Department of Tumor Biology, Karolinska Institutet, Stockholm, Sweden.
Boldog
F
F
Thompson
T
T
Scanlon
D
D
Winberg
G
G
Marcsek
Z
Z
Erlandsson
R
R
Stanbridge
E J
EJ
Klein
G
G
Sümegi
J
J
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D008040:Genetic Linkage; D015698:Genomic Library; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology; D007621:Karyotyping; D008677:Metaphase; D051379:Mice; D015183:Restriction Mapping
D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases