Improved allelic differentiation using sequence-specific oligonucleotide hybridization incorporating an additional base-analogue mismatch.
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.
Burgner
David
D
Department of Paediatrics, University of Oxford, Oxford, UK.
D'Amato
Mauro
M
Kwiatkowski
Dominic P
DP
Loakes
David
D
D000483:Alleles; D020137:Base Pair Mismatch; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011758:Pyrroles / Q000737:chemistry*; D044366:Transition Temperature
D015345:Oligonucleotide Probes; D011758:Pyrroles; C094911:3-nitropyrrole