Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization

Citation
Pk. Rogan et al., Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization, GENOME RES, 11(6), 2001, pp. 1086-1094
Citations number
28
Categorie Soggetti
Molecular Biology & Genetics
Journal title
GENOME RESEARCH
ISSN journal
10889051 → ACNP
Volume
11
Issue
6
Year of publication
2001
Pages
1086 - 1094
Database
ISI
SICI code
1088-9051(200106)11:6<1086:SDOSGD>2.0.ZU;2-9
Abstract
Chromosomal rearrangements are frequently monitored by Fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of con tiguous genomic intervals that are often distant From disease loci. We deve loped smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computati onal sequence analysis of similar to 100-kb genomic sequences. ScFISH probe s are produced by long PCR, then purified, labeled, and hybridized individu ally or in combination to human chromosomes. Preannealing or blocking with unlabeled, repetitive DNA is unnecessary, as scFISH probes lack repetitive DNA sequences. The hybridization results are analogous to conventional FISH , except that shorter probes can be readily visualized. Combinations of pro bes from the same region gave single hybridization signals on metaphase chr omosomes. ScFISH probes are produced directly from genomic DNA, and thus mo re quickly than by recombinant DNA techniques. We developed single-copy pro bes for three chromosomal regions-the CDC2L1 (chromosome 1p36), MAGEL2 (chr omosome 15q11.2), and HIRA (chromosome 22q11.2) genes-and show their utilit y for FISH. The smallest probe tested was 2290 bp in length. To assess the potential utility of scFISH For high-resolution analysis, we determined chr omosomal distributions of such probes. Single-copy intervals of this length or greater are separated by an average of 29.2 and 22.3 kb on chromosomes 21 and 22, respectively. This indicates that abnormalities seen on metaphas e chromosomes could be characterized with scFISH probes at a resolution gre ater than previously possible.