Amplification and detection method
View Patent ↗The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.
1. A method of amplifying and detecting the presence of a target nucleic acid in a reaction mixture, the method comprising the steps of:
(a) performing a transcription-based amplification reaction to produce a transcription product having an RNA sequence contained within or complementary to a RNA or equivalent DNA sequence of the target nucleic acid; and
(b) detecting the transcription product as an indication of the presence of the target nucleic acid in the reaction mixture, the transcription product forming a hybridization complex with a target binding region of a probe molecule, wherein the target binding region comprises at least one 2′-O-methyl ribonucleotide.
2. The method of claim 1 , wherein the target binding region comprising a cluster of at least 4 contiguous 2′-O-methyl ribonucleotides.
3. The method of claim 1 , wherein the target binding region consists of 2′-O-methyl ribonucleotides.
4. The method of claim 1 , wherein the probe molecules comprises a conjugate molecule.
5. The method of claim 2 , wherein the probe molecules comprises a conjugate molecule joined to the target binding region at a site located within the cluster of 2′-O-methyl ribonucleotides.
6. The method of claim 1 , wherein the probe molecules is from 10 to and 100 nucleotide bases in length.
7. The method of claim 1 , wherein the probe molecules is from 10 to 16 nucleotide bases in length.
8. The method of claim 1 , wherein the label is a chemiluminescent or a fluorescent agent.
9. The method of claim 1 , wherein the probe molecules comprise self-complementary regions which include the at least one 2′-O-methyl ribonucleotide.
10. The method of claim 1 , wherein the transcription product comprises a structured region, and wherein the target binding region forms a hybridization complex with the structured region in step (b).
11. The method of claim 1 further comprising the step of quantifying the amount of target nucleic acid present in the reaction mixture.
12. The method of claim 1 , wherein the amplification reaction is a transcription-mediated amplification reaction.