Genome Research · 2003 · 132 citations · 1 references
GeneticsGenetic EpidemiologyHuman PolymorphismMolecular BiologyMolecular GeneticsGenomicsHolliday JunctionGenome-wide Association StudyGenetic AnalysisGenotype-phenotype AssociationNew MechanismPublic HealthMolecular DiagnosticsMolecular Biological MethodSnp ScoringAllelic VariantNucleic Acid AmplificationAllelic DiscriminationPcr PrimersSystems BiologyMedicine
We report here a new mechanism for allelic discrimination--allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.
1