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Sequence-specific oligonucleotide hybridization (SSOH, 'dot-blotting') is a widely employed method of typing single nucleotide polymorphisms (SNPs), but it is often compromised by lack of allelic differentiation. We describe a novel improvement to SSOH that incorporates an additional mismatch into the oligonucleotide probe using the universal base analogue 3-nitropyrrole. This method greatly increases allelic differentiation compared to standard SSOH where oligonucleotides contain only SNP-defining base changes. Moreover, stringency of the hybridisation is predictably maintained over a wide range of temperatures, which can be calculated empirically, thus facilitating the genotyping of multiple SNPs using similar conditions. This improved method increases the usefulness of hybridisation-based methods of rapid genotyping of SNPs and may have implications for array methodologies.

Original publication

DOI

10.1081/ncn-120039216

Type

Journal article

Journal

Nucleosides, nucleotides & nucleic acids

Publication Date

05/2004

Volume

23

Pages

755 - 765

Addresses

Department of Paediatrics, University of Oxford, Oxford, UK.

Keywords

Pyrroles, Oligonucleotide Probes, Nucleic Acid Hybridization, Base Pair Mismatch, Polymorphism, Single Nucleotide, Alleles, Transition Temperature