Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis.
Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.
Basile
Andrea
A
Institute of Chemistry of Molecular Recognition, C.N.R., Via Mario 9, I-20131 Milan, Italy.
Giuliani
Andrea
A
Pirri
Giovanna
G
Chiari
Marcella
M
D020029:Base Pairing; D002232:Carbocyanines; D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D016296:Mutagenesis; D020135:Peptide Nucleic Acids
D002232:Carbocyanines; D015342:DNA Probes; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; C085321:cyanine dye 5; D004247:DNA