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
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.