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

Electrophoresis
Andrea BasileMarcella Chiari

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.

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Citations

Apr 30, 2004·Cell Research·Gan Wang, Xiaoxin S Xu
Aug 17, 2011·International Journal of Molecular Sciences·Shohei KanedaTeruo Fujii
May 16, 2015·Molecular Biotechnology·Grzegorz MachnikBogusław Okopień
Jan 17, 2003·Expert Review of Molecular Diagnostics·Gabor L Igloi
Nov 2, 2007·Chemical Reviews·Karel Kleparník, Petr Bocek
Oct 14, 2004·Journal of the American Chemical Society·Karin M BalssMichael J Tarlov

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