Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring.
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.
Yang
Qinghong
Q
FreshGene, Inc., Concord, California 94520, USA. qinghong@freshgene.com
Lishanski
Alla
A
Yang
Wendy
W
Hatcher
Sandra
S
Seet
Henrietta
H
Gregg
Jeffrey P
JP
D000483:Alleles; D020137:Base Pair Mismatch / Q000235:genetics; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics; D030541:Databases, Genetic; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004706:Endodeoxyribonucleases / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D020862:GC Rich Sequence / Q000235:genetics; D005838:Genotype; D008957:Models, Genetic; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011995:Recombination, Genetic; D012641:Selection, Genetic
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases; C071059:Holliday junction DNA helicase, E coli; D004265:DNA Helicases