An efficient chemical method to generate repetitive sequences depleted DNA probes

We describe an efficient method to remove repetitive sequences from DNA of microdissected whole chromosomes, chromosome arms, locus specific probes, and specific bands. The chemical approach described removes human repetitive DNA sequences from a source DNA, eliminating the need for blocking such sequences when the source DNA used as a probe is hybridized with a target DNA. It employs subtracting hybridized biotin‐labeled repetitive‐sequence DNA complex with phenol and chloroform after incubation of hybridized products with avidin. The method produces unique products that are formed after such repetitive sequences have been removed from the DNA. We have applied our newly designed subtraction strategy to microdissected chromosomes in generating whole chromosome painting (e.g., chromosome 4), chromosome arm (e.g., 1q and 15q), and band (e.g., 3q26) specific probes, and we have demonstrated its utility using flow‐sorted chromosome. FISH analyses using these probes show consistent strong signals with no cross‐hybridization, and optimal hybridization results can be obtained relatively quickly. © 2006 Wiley‐Liss, Inc.

[1]  D. Ward,et al.  Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries , 1988, Human Genetics.

[2]  P. Meltzer,et al.  Coverage of chromosome 6 by chromosome microdissection: generation of 14 subregion-specific probes , 1995, Human Genetics.

[3]  T. Cremer,et al.  Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography , 1997, Human Genetics.

[4]  J. Clapp,et al.  The use of selective enrichment for the isolation of species-specific DNA probes for insects. , 1996, Methods in molecular biology.

[5]  M. Bittner,et al.  Chromosome arm painting probes , 1996, Nature Genetics.

[6]  P S Meltzer,et al.  Generation of band-specific painting probes from a single microdissected chromosome. , 1993, Human molecular genetics.

[7]  P. Meltzer,et al.  Rapid generation of region specific probes by chromosome microdissection and their application , 1992, Nature Genetics.

[8]  W. Kuo,et al.  Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes. , 1991, Genomics.

[9]  J. Frézal,et al.  Report of the committee on clinical disorders, chromosome aberrations and uniparental disomy , 1991 .

[10]  J. Trent,et al.  Report of the committee on chromosome changes in neoplasia , 1990 .

[11]  A. Sandberg.  The chromosomes in human cancer and leukemia , 1990 .

[12]  D. Pinkel,et al.  Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Singer.  Highly repeated sequences in mammalian genomes. , 1982, International review of cytology.

[14]  Olufunmilayo I. Olopade,et al.  The Chromosomes in Human Cancer and Leukemia , 1981 .

[15]  J. Yunis,et al.  Retinoblastoma and subband deletion of chromosome 13. , 1978, American journal of diseases of children.

[16]  J. Yunis.  Trisomy for the distal end of the short arm of chromosome 3: a syndrome. , 1978, American journal of diseases of children.

[17]  G. Klein,et al.  Characteristic chromosomal abnormalities in biopsies and lymphoid‐cell lines from patients with burkitt and non‐burkitt lymphomas , 1976, International journal of cancer.