Improved allelic differentiation using sequence-specific oligonucleotide hybridization incorporating an additional base-analogue mismatch

David Burgner, Mauro D'Amato, Dominic P. Kwiatkowski, David Loakes

Research output: Contribution to journalArticleResearchpeer-review

8 Citations (Scopus)

Abstract

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 languageEnglish
Pages (from-to)755-765
Number of pages11
JournalNucleosides, Nucleotides and Nucleic Acids
Volume23
Issue number5
DOIs
Publication statusPublished - 27 Jul 2004
Externally publishedYes

Keywords

  • Base mismatch
  • Dot-blotting
  • Sequence-specific oligonucleotide hybridization
  • Universal base analogue

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