Cosmid assembly and anchoring to human chromosome 21.
A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.
Soeda
E
E
RIKEN Gene Bank, Institute of Physical and Chemical Research, Tsukuba Science City, Japan.
Hou
D X
DX
Osoegawa
K
K
Atsuchi
Y
Y
Yamagata
T
T
Shimokawa
T
T
Kishida
H
H
Soeda
E
E
Okano
S
S
Chumakov
I
I
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D002891:Chromosomes, Human, Pair 21; D003360:Cosmids; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D016172:DNA Fingerprinting; D017931:DNA Primers; D005456:Fluorescent Dyes; D015723:Gene Library; D006801:Humans; D006822:Hybrid Cells; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D005456:Fluorescent Dyes; D004247:DNA