Genotyping single nucleotide polymorphisms directly from genomic DNA by invasive cleavage reaction on microspheres.

Here we report proof-of-principle for a microsphere-based genotyping assay that detects single nucleotide polymorphisms (SNPs) directly from human genomic DNA samples. This assay is based on a structure-specific cleavage reaction that achieves single base discrimination with a 5'-nuclease which recognizes a tripartite substrate formed upon hybridization of target DNA with probe and upstream oligonucleotides. The assay is simple with two easy steps: a cleavage reaction, which generates fluorescent signal on microsphere surfaces, followed by flow cytometry analysis of the microspheres. Genomic DNA samples were genotyped for the SNP in the Apolipoprotein E gene at amino acid position 158. The assay successfully scored wild type, heterozygous and homozygous mutants. To our knowledge, this is the first report of a solid-support assay for detection of SNPs directly from genomic DNA without PCR amplification of the target.

A surface invasive cleavage assay for highly parallel SNP analysis.

The structure-specific invasive cleavage of single-stranded DNA by 5' nucleases is a useful means for sensitive detection of single-nucleotide polymorphisms or SNPs. The solution-phase invasive cleavage reaction has sufficient sensitivity for direct detection of as few as 600 target molecules with no prior target amplification. One approach to the parallelization of SNP analysis is to adapt the invasive cleavage reaction to an addressed array format. Two surface invasive cleavage reaction strategies were designed and tested using the polymorphic site in codon 158 of the human ApoE gene as a model system, with a synthetic oligonucleotide as target. The upstream oligonucleotide, which is required for the invasive cleavage reaction, was either added in solution (strategy 1), or co-immobilized on the surface along with the probe oligonucleotide (strategy 2). Both strategies showed target-concentration and time-dependent amplification of signal. Parameters that govern the rate of the surface-invasive cleavage reactions are discussed.

Imaging and analysis of immobilized particle arrays.

An automated imaging system was developed to quantify fluorescence signals from particles immobilized on hydrogel-coated slides. Arrays of submicrometer-diameter particles were printed with up to 600 particles/spot. The slides were read under 20x magnification without cover slips. Software was written to image individual spots and measure the median particle fluorescence in each spot. To locate array spots, an alignment program made use of two fiducial grids of fluorescent reference particles at either end of the slide. Focusing was adjusted locally using spots of reference particles located at the centers of focusing neighborhoods. The response was linear across a two-decade range, and the precision of readings was better than 5% down to approximately 1000 fluors/particle. Exposure times varied with signal intensity, reaching 1 s at the lowest levels of fluorescence. Data demonstrate feasibility for measuring fluorescence from immobilized particle arrays on an automated microscope with accuracy and precision similar to fluorescence measurements of microparticles with a flow cytometer. This work provides automation of imaging and analysis procedures necessary for development of immobilized particle arrays as an analytical platform that combines advantageous features of both planar and suspension arrays.

Immobilized particle arrays: coalescence of planar- and suspension-array technologies.

Combining positive attributes of planar arrays and suspension arrays, immobilized particle arrays offer a new format in which immobilized submicrometer particles are arrayed on hydrogel-coated slides, providing 100+ assay replicates within each spot. This research describes how to prepare immobilized protein arrays and how to assay the binding of labeled target molecules to the arrayed capture probes. The assay system exhibits an intrinsic dynamic range of two to three decades, with coefficients of variation from 5 to 10%. For antibody-antigen binding, target capture appears to be reaction rate limited. For labeled antibody binding to antigen on the immobilized particles, the detection limit is approximately 0.5 ng/mL. When antibodies on the immobilized particles exhibit multivalent binding of target molecules, the detection limit is approximately 0.01 ng/mL. For protein arrays, potential advantages of this format are improved coating of the capture reagent, an increased number of options for protein presentation, reduced mass transport effects, and higher density multiplexing.

Structure-specific DNA cleavage on surfaces.

The structure-specific invasive cleavage reaction is a useful means for sensitive and specific detection of single nucleotide polymorphisms, or SNPs, directly from genomic DNA without a need for prior target amplification. A new approach integrating this invasive cleavage assay and surface DNA array technology has been developed for potentially large-scale SNP scoring in a parallel format. Two surface invasive cleavage reaction strategies were designed and implemented for a model SNP system in codon 158 of the human ApoE gene. The upstream oligonucleotide, which is required for the invasive cleavage reaction, is either co-immobilized on the surface along with the probe oligonucleotide or alternatively added in solution. The ability of this approach to unambiguously discriminate a single base difference was demonstrated using PCR-amplified human genomic DNA. A theoretical model relating the surface fluorescence intensity to the progress of the invasive cleavage reaction was developed and agreed well with experimental results.