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

Rolling circle amplification (RCA) is a surface-anchored DNA replication re action that can be exploited to visualize single molecular recognition even ts. Here we report the use of RCA to visualize target DNA sequences as smal l as 50 nts in peripheral blood lymphocytes or in stretched DNA fibers. Thr ee unique target sequences within the cystic fibrosis transmembrane conduct ance regulator gene could be detected simultaneously in interphase nuclei, and could be ordered in a linear map in stretched DNA, Allele-discriminatin g oligonucleotide probes in conjunction with RCA also were used to discrimi nate wild-type and mutant alleles in the cystic fibrosis transmembrane cond uctance 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 mult icolor fluorescence imaging to detect single nucleotide changes in DNA with in 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.