Post-synthesis labeling of nucleic acids, assays using nucleic acids that are labeled post-synthetically, single nucleotide polymorphism detection, and associated compounds and microarrays
View Patent ↗An assay is provided for nucleic acids that can be post-synthetically labeled, wherein modified nucleoside triphosphates are used that are more efficiently and specifically incorporated during nucleic acid synthesis than labeled nucleoside triphosphates. In a preferred embodiment, nucleoside α-thiotriphosphates are utilized. Maleimide or iodoacetamide conjugating moieties can be attached post-synthetically. The conjugating moieties may include a reporter group. Also disclosed are new methods for detecting single nucleotide polymorphism.
1. In a nucleic acid expression assay, comprising (1) performing in vitro transcription of target nucleic acid template to generate cRNA, (2) contacting said cRNA with a probe immobilized on a microarray under conditions that allow hybridization between said cRNA and said probe, and (3) detecting hybridized cRNA to assay expression of said target nucleic acid template, the improvement comprising, (a) during said performing step, incorporating in said cRNA at least one phosphorothioate moiety; and (b) labeling said cRNA by conjugating a reporter molecule to said phosphorothioate moiety wherein said labeling step comprises reacting said target nucleic acid with a conjugating moiety that specifically reacts with said phosphorothioate moiety, followed by reaction with a labeling moiety that specifically reacts with said conjugating moiety.
2. The method of claim 1 , wherein said labeling step follows said contacting step.
3. The method of claim 1 , wherein said reporter molecule has an electrophilic moiety.
4. The method of claim 1 , wherein said conjugating moiety is an electrophilic moiety.
5. The method of claim 3 , wherein said electrophilic moiety is selected from the group consisting of a maleimide and an iodoacetamide.
6. The method of claim 1 , wherein said reporter molecule is selected from the group consisting of a fluorophore, a redox moiety, and an electrochemically active agent.
7. The method of claim 1 , wherein said reporter molecule is selected from the group consisting of TMR-maleimide, TMR-iodoacetamide and ALEXAFLUOR-maleimide.
8. The method of claim 1 , wherein said cRNA has at least three different thio ribonucleotides, said thio ribonucleotides being selected from the group consisting of an adenosine thiophosphate, a cytidine thiophosphate, a guanosine thiophosphate, a thymidine thiophosphate, and a uridine thiophosphate.