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.

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis / A. Basile, A. Giuliani, G. Pirri, M. Chiari. - In: ELECTROPHORESIS. - ISSN 0173-0835. - 23:6(2002 Mar), pp. 926-929.

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis

A. Basile
Primo
;
2002

Abstract

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.
Capillary electrophoresis; Hybridization; Mutation detection; Nucleic acid; Peptide nucleic acid
Settore MED/46 - Scienze Tecniche di Medicina di Laboratorio
mar-2002
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/19816
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