DNA glycosylase/lyase and AP endonuclease substrates
AssigneeAlere San Diego, Inc.
InventorsOlaf Piepenburg, Niall A. Armes
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Abstract
A new class of nucleic acid substrates for AP endonucleases and members of the glycosylase/lyase family of enzymes is described. Representatives of each family, the enzymes Nfo and fpg, respectively, cleave nucleic acid backbones at positions in which a base has been replaced by a linker to which a variety of label moieties may be attached. The use of these synthetic substrates embedded within oligonucleotides is of utility in a number of applications.
Claims28
A process of detecting the presence or absence of a target nucleic acid, comprising the steps of:
- (a) contacting an oligonucleotide probe to a target nucleic acid, thereby forming a complex between the probe and the target nucleic acid, wherein the probe comprises a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms wherein n is 1-8;
- (b) contacting the probe/nucleic acid complex with a nuclease selected from the group consisting of AP endonuclease, DNA glycosylase/lyase and DNA glycosylase, wherein the nuclease can excise the linker from the complex and/or specifically cleave the probe at the dR-O—[C]n nucleotide;
- and
- (c) detecting whether such excision or cleavage has occurred.
A process of cleaving an oligonucleotide in a complex with a nucleic acid, which comprises contacting the oligonucleotide/nucleic acid complex with a nuclease selected from the group consisting of AP endonuclease, DNA glycosylase/lyase and DNA glycosylase, wherein the oligonucleotide contains a dR-O—[C]n nucleotide that lacks a base and has a sugar with a carbon at a 1′ position, and wherein the carbon at the 1′ position is covalently linked through an oxygen atom to a carbon atom of a linker containing n carbon atoms wherein n is 1-8;
- and wherein the nuclease specifically cleaves the probe at the dR-O—[C]n nucleotide.
Dependencies are inferred from the claim text.