Structure-Specific DNA Cleavage on Surfaces

Abstract

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.

Invasive cleavage reactions on DNA‐modified diamond surfaces

“…target, for example, 100 amole, which is equivalent to the amount of genomic DNA extracted from ϳ10 mL whole blood. 39 However, the detection sensitivity in our previous study 27,28 was limited by the long-term degradation of the DNA-modified surfaces at elevated temperature. At relatively high target concentration, a very short reaction time can generate enough signal increase for detection (Figure 3), whereas at low target concentration, as is the case for the analysis of genomic DNA samples, much longer reaction times are needed as the accumulation of the cleavage product, or the signal generation increases linearly as a function of time.…”

“…26 We previously proposed a very powerful approach for largescale SNP genotyping on surface DNA microarrays using this invasive cleavage assay; we demonstrated the feasibility of performing such reaction on DNAmodified gold surfaces using PCR-amplified genomic DNA as target. 27,28 However, the detection sensitivity of this new approach suffered from instability of the attachment chemistry of DNA oligonucleotides to the solid substrate, particularly at elevated temperatures.…”

Carbon Substrates: A Stable Foundation for Biomolecular Arrays

“…Increasing the physical distance between a fluorescent dye and a quencher causes an increase in the fluorescence intensity of the dye; the quenching mechanism is a result of fluorescence resonance energy transfer (FRET) (29). FRET-based readouts have been used to screen hybridization of biological samples to molecular beacons immobilized on the surface (i.e., an aptamer array) (30–33) and in assays that rely on the enzymatic cleavage of immobilized oligonucleotides (34, 35). …”

Covalent attachment of shape-restricted DNA molecules on amine-functionalized Si(111) surface

“…Because the substrates used were transparent in a visible optical range, the majority of the investigations are focused on optical methods of detection. Gold substrates [11][12][13][14][15][16][17][18] were also widely used to immobilize biomolecules. However, there is a need to enhance the stability of the interface systems.…”