New strategy for mapping the human genome based on a novel procedure for construction of jumping libraries.
A novel procedure for construction of jumping libraries is described. The essential features of this procedure are as follows: (1) two diphasmid vectors (lambda SK17 and lambda SK22) are simultaneously used in the library construction to improve representativity, (2) a partial filling-in reaction is used to eliminate cloning of artifactual jumping clones and to obviate the need for a selectable marker. The procedure has been used to construct a representative human NotI jumping library (220,000 independent recombinant clones) from the lymphoblastoid cell line CBMI-Ral-STO, which features a low level of methylation of its resident EBV genomes. A human chromosome 3-specific NotI jumping library (500,000 independent recombinant clones) from the human chromosome 3 x mouse hybrid cell line MCH 903.1 has also been constructed. Of these recombinant clones 50-80% represent jumps to the neighboring cleavable NotI site. With our previously published method for construction of linking libraries this procedure makes a new genome mapping strategy feasible. This strategy includes the determination of tagging sequences adjacent to NotI sites in random linking and jumping clones. Special features of the lambda SK17 and lambda SK22 vectors facilitate such sequencing. The STS (sequence tagged site) information obtained can be assembled by computer into a map representing the linear order of the NotI sites for a chromosome or for the entire genome. The computerized mapping data can be used to retrieve clones near a region of interest. The corresponding clones can be obtained from the panel of original clones, or necessary probes can be made from genomic DNA by PCR.
Zabarovsky
E R
ER
Department of Tumor Biology, Karolinska Institutet, Stockholm, Sweden.
Boldog
F
F
Erlandsson
R
R
Kashuba
V I
VI
Allikmets
R L
RL
Marcsek
Z
Z
Kisselev
L L
LL
Stanbridge
E
E
Klein
G
G
Sumegi
J
J
D000818:Animals; D002874:Chromosome Mapping / Q000379:methods*; D002893:Chromosomes, Human, Pair 3; D003001:Cloning, Molecular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D015723:Gene Library; D016147:Genes, Tumor Suppressor; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D014407:Tumor Cells, Cultured
D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases