Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions

AUTOR(ES)

Wilkins Stevens, Priscilla

FONTE

Oxford University Press

RESUMO

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5′-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Documentos Relacionados

• Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.
• Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres
• High-Throughput Genotyping with Single Nucleotide Polymorphisms
• Single Nucleotide Polymorphisms Associated With Rat Expressed Sequences
• Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER)