The generation of DNA probes to chromosome 11q23 by Alu PCR on small numbers of flow-sorted 22q- derivative chromosomes.
A strategy for the isolation of DNA probes from small numbers of flow-sorted human chromosomes has been developed. A lymphoblastoid cell line carrying the 22q- derivative chromosome product of the constitutional t(11;22) translocation was used as the source of chromosomes. Synthetic oligonucleotide primers, based on the consensus Alu sequence, were used to amplify inter-Alu sequence from 500 flow-sorted 22q- derivative chromosomes. The amplified sequences were cloned into a plasmid vector by blunt-end ligation, yielding clones with inserts in the range of 400 to 1000 bp. Approximately 70% of these clones hybridized to human DNA as single-copy probes. To identify clones derived from chromosome 11, the library was screened with a heterogeneous probe prepared by Alu-PCR amplification from the DNA of a somatic cell hybrid containing one homology of chromosome 11. All the positive clones found were mapped to within the q23-q25 region of chromosome 11 known to be translocated onto the 22q- derivative chromosome. Further mapping studies showed that most of these probes (7/8) lay between the breakpoints for the t(4;11) translocation of acute lymphocytic leukemia and the t(11;22) of Ewing sarcoma. Thus, the use of Alu-PCR on the small derivative chromosome 22q- has provided a greatly enriched source of probes to region 11q23, a part of the genome that is currently of great interest. This approach will be particularly appropriate to small numbers of chromosomes when high specificity rather than total representation is required.
Cotter
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ICRF Department of Medical Oncology, St. Bartholomew's Hospital, London, England.
Das
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Douek
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Carter
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Young
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D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11; D003001:Cloning, Molecular; D016384:Consensus Sequence; D015342:DNA Probes; D005434:Flow Cytometry; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D014178:Translocation, Genetic / Q000235:genetics
D015342:DNA Probes; D009838:Oligodeoxyribonucleotides