Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification

Abstract

Rolling circle amplification (RCA) is a surface-anchored DNA replication reaction that can be exploited to visualize single molecular recognition events. Here we report the use of RCA to visualize target DNA sequences as small as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Three unique target sequences within the cystic fibrosis transmembrane conductance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA. Allele-discriminating oligonucleotide probes in conjunction with RCA also were used to discriminate wild-type and mutant alleles in the cystic fibrosis transmembrane conductance regulator, p53, BRCA-1, and Gorlin syndrome genes in the nuclei of cultured cells or in DNA fibers. These observations demonstrate that signal amplification by RCA can be coupled to nucleic acid hybridization and multicolor fluorescence imaging to detect single nucleotide changes in DNA within a cytological context or in single DNA molecules. This provides a means for direct physical haplotyping and the analysis of somatic mutations on a cell-by-cell basis.

Figure 1

Schematic for detecting small target sequences (A) or point mutations (B) using RCA. See text for description.

Figure 2

Visualization of ODN probes hybridized to 50-mer target sequences in the CFTR gene in interphase nuclei and DNA fibers. (A) RCA detection of probes targeted to the G542X locus (FITC), the Δ508 locus (Cy-3), and the M1101K locus (Cy5) in normal human lymphocytes. Merged images (com) show that signals from all three loci colocalize. (B) Hybridization of the Δ508 locus probe to nuclei of HeLa cells. (C) Cohybridization of two PAC clones (extended green and red signals) with Δ508 (yellow), G542X (green), and M1101K (white) oligomer probes. Eight patterns (a–h) of hybridization are produced (see text).

Figure 3

RCA detection of wt and mu alleles at the G542X locus of the CFTR gene. (A) Nuclei from wt, homozygous mu, and heterozygous cells in G₁ phase (Upper) or G₂ phase (Lower) of cell cycle. (B) Discrimination of wt and mu alleles of the G542X locus on stretched DNA fibers.

Figure 4

RCA detection of wt and mu alleles at the A13073C locus of the p53 gene, the 5382C ins locus of the BRCA gene, and the C3383A locus of the patched (Gorlin syndrome) gene.