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Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization

By Heather Perry-O’Keefe, Xian-Wei Yao, James M. Coull, Martin Fuchs and Michael Egholm

Abstract

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis

Topics: Biological Sciences
Publisher: The National Academy of Sciences of the USA
Year: 1996
OAI identifier: oai:pubmedcentral.nih.gov:26193
Provided by: PubMed Central
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