A straightforward approach to isolate DNA sequences with potential linkage to the retinoblastoma locus

Abstract

From a human-Chinese hamster somatic cell hybrid a clone was derived containing chromosome 13 in duplicate as its only human material. This clone was used to construct a human chromosome 13-specific recombinant DNA-library. Overlapping Sau3AI DNA sequences (11.9-17.2 kb) from the cell hybrid were inserted into the lambda phage vector EMBL4. From eleven recombinants having a human insert thirteen putative unique DNA sequences were isolated and cloned into the plasmid vector pBR329. A human-mouse hybrid containing a human chromosome 13 with a deletion of 13q14 and lacking its undeleted homologue was constructed to be used in a selection procedure for DNA sequences belonging to band q14. Three probes originating from two different phages were assigned to 13q14 because they did not hybridise to DNA from this cell hybrid. One of these 13q14 probes detects a low frequency (2/44) MspI restriction fragment length polymorphism. The probes are now being used for screening a cosmid library to find adjacent polymorphic sequences with a RFLP information content suitable for application in the diagnosis of hereditary retinoblastoma.

A High-Resolution Physical Map of Human Chromosome 21p Using Yeast Artificial Chromosomes

“…) were detected using PCR on human genomic DNA of CEPH families 1333, 1334, 1347, or 7 unrelated individuals, and a set of somatic cell hybrids containing acrocentric chromosomes. GB3 contains chromosome 13 (Scheffer et al 1986), HDm-5 (Lugo et al 1987) and WegrothB3 (Geurts van Kessel et al 1983) chromosome 14, HorlI chromosome 15 (Heisterkamp et al 1982), and Wegroth D2 chromosome 22 (Geurts van Kessel et al 1983). The PCR reaction was carried out in a total volume of 10 µl containing ∼100 ng template DNA, 0.1 unit of Taq DNA polymerase, 5 pmoles of each primer, one of which was ␥-32 P end-labeled, 100 µM dNTP, and 1.0 mM MgCl 2 .…”

Construction of the physical map for three loci in chromosome band 13q14: comparison to the genetic map.

“…The disparity in the gene order determined by the two approaches led to a reexamination of the linkage analysis that resulted in the construction of the multilocus map (21) and raises questions about confidence levels for gene orders in three-locus and four-locus linkage analysis. The confidence levels associated with log-likelihood differences and odds ratios have not been determined.…”

“…PFGE has been used previously to establish a long-range restriction map surrounding the RBI locus and to localize the breakpoints of three retinoblastoma-associated translocations within the RBI gene (18). This map is being extended in both directions by making use of cloned DNA probes derived from several chromosome 13-specific libraries (19)(20)(21) and a panel of cell lines carrying overlapping deletions of chromosome 13.…”

Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes

“…Cell lines C121 (a kind gift of Dr. J. Goodfellow), GF7 (26), CY18 (2), and 72532X-6 (29) contain only one human chromosome each, Nos. 7, 13, 16, and 21, respectively.…”