Restriction mapping in nanofluidic devices.

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg2+ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of approximately 1.

Invasive cleavage reactions on DNA-modified diamond surfaces.

Recently developed DNA-modified diamond surfaces exhibit excellent chemical stability to high-temperature incubations in biological buffers. The stability of these surfaces is substantially greater than that of gold or silicon surfaces, using similar surface attachment chemistry. The DNA molecules attached to the diamond surfaces are accessible to enzymes and can be modified in surface enzymatic reactions.

Surface amplification of invasive cleavage products.

A major focus of current efforts in genomics is to elucidate the genetic variations extent within the human population, and to study the effects of these variations upon the human system. The most common type of genetic variations are the single nucleotide polymorphisms (SNPs), which occur every 500-1000 nt in the genome. Large-scale population association studies to study the biological or medical significance of such variations may require the analysis of hundreds of thousands of SNPs on thousands of individuals.

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.

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.