Genetics

Hitting Singles

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Science  13 Apr 2001:
Vol. 292, Issue 5515, pp. 173
DOI: 10.1126/science.292.5515.173c

Fluorescence in situ hybridization has been instrumental in visualizing discrete regions (as small as several hundred nucleotides) of chromosomes in single cells, and, of course, DNA sequencing and standard cloning techniques have been used to identify single nucleotide changes associated with genetic polymorphisms and disorders.

Now, Zhong et al. have developed a protocol in which single base changes can be visualized in single cells. As a test system, they focused on the G542X locus within the cystic fibrosis (CFTR) gene as it was represented in wild-type peripheral blood lymphocytes and in two mutant cell lines homozygous and heterozygous for this locus. Rolling circle DNA amplification (RCA) and specific oligonucleotide probes revealed two green (FITC) nuclear signals in wild-type cells, two red (Cy3) signals in homozygous cells, and one of each color in heterozygous cells. These results were confirmed in fiber DNA analyses with the addition of two flanking loci (Δ508 and M1101K) providing both color and relative distance markers. Wild-type and mutant alleles in the p53, BRCA, and patched genes also were visualized in single cells by RCA. — GJC

Proc. Natl. Acad. Sci. U.S.A.98, 3940 (2001).

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