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.
|Titolo:||Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis|
|Autori interni:||BASILE, ANDREA (Primo)|
|Parole Chiave:||Capillary electrophoresis; Hybridization; Mutation detection; Nucleic acid; Peptide nucleic acid|
|Settore Scientifico Disciplinare:||Settore MED/46 - Scienze Tecniche di Medicina di Laboratorio|
|Data di pubblicazione:||mar-2002|
|Digital Object Identifier (DOI):||10.1002/1522-2683(200203)23:6<926::AID-ELPS926>3.0.CO;2-J|
|Appare nelle tipologie:||01 - Articolo su periodico|