Nucleic acid amplification using a reversibly modified oligonucleotide

• US 9,045,522 B2
• Filed: 11/20/2012
• Issued: 06/02/2015
• Est. Priority Date: 07/31/2006
• Status: Expired due to Fees

• Current Assignee: Suzhou Nuhigh Biotechnologies Co., Ltd.
• Original Assignee: Wanli Bi
• Inventors: Bi, Wanli
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/682,664
• Publication Number:
  US 20130122507A1
• Time in Patent Office: 924 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The present invention provides a method for amplification of a target nucleic acid sequence or signal, wherein an amplification reaction mixture is used which contains at least one reversibly modified oligonucleotide having a non-hydroxyl group 3′ end which can be converted into a hydroxyl 3′ end upon exposure to a chemical and/or irradiation and/or a range of temperature. The present invention also provides a reversibly modified oligonucleotide as described above, and a nucleic acid amplification reaction mixture and kit comprising such an oligonucleotide.

Claims

1. A method for regenerating a 3′

hydroxyl group, the method comprising;

using a mixture comprising at least one reversibly modified oligonucleotide having a non-hydroxyl group 3′

end which can be converted into a 3′

hydroxyl group, wherein the oligonucleotide has a carboxylic acid ester group at its 3′

end;

exposing the mixture to a first chemical and a first range of temperature, wherein the first chemical and the first range of temperatures regenerate the 3′

hydroxyl group, wherein the first chemical is an amine;

wherein the amine is selected from the group consisting of methylamine, ethylenediamine, and triethylamine, and

regenerating the 3′

hydroxyl group of the at least one reversibly modified oligonucleotide having a non-hydroxyl 3′

end.

2. The method of claim 1, wherein the amine is methylamine.

3. The method of claim 1, wherein the amine is ethylenediamine.

4. The method of claim 1, wherein the amine is triethylamine.

5. The method of claim 1, wherein the carboxylic acid ester is maleic acid ester, and the first chemical is methylamine.

6. The method of claim 1, wherein the carboxylic acid ester is maleic acid ester, and the first chemical is ethylenediamine.

7. The method of claim 1, wherein the carboxylic acid ester is maleic acid ester, and the first chemical is triethylamine.

8. The method of claim 1, further comprising:

contacting a sample suspected of containing a target nucleic acid with the mixture comprising the at least one reversibly modified oligonucleotide with a regenerated 3′

hydroxyl group; and

conducting an amplification reaction.

9. The method of claim 8, wherein the target nucleic acid is ribonucleic acid, and wherein reverse transcription of the ribonucleic acid is conducted prior to the amplification reaction.

10. The method of claim 8, wherein amplification of the ribonucleic acid (i) is a one-step RT-PCR process with a two-enzyme system, in which at least a reverse transcriptase and a thermostable DNA polymerase is used, or (ii) is a one-step RT-PCR process with a one-enzyme system, in which only one enzyme is used which functions as both a reverse transcriptase and a DNA polymerase.

11. The method of claim 8, wherein the amplification reaction is selected from the group consisting of invader assay, polymerase chain reaction, ligase chain reaction, rolling circle amplification, strand displacement amplification, transcription mediated amplification, nucleic acid sequence based amplification, self-sustained sequence replication, single primer isothermal amplification, exponential single primer isothermal amplification, and loop mediated amplification.

12. The method of claim 1, wherein the mixture further comprises at least one second reversibly modified oligonucleotide, wherein the at least one second reversibly modified oligonucleotide has a non-hydroxyl group 3′

end which can be converted into a hydroxyl 3′

end, and wherein the method further comprises exposing the mixture to a second chemical and a second range of temperatures and regenerating the hydroxyl 3′

end of the at least one second reversibly modified oligonucleotide having a non-hydroxyl 3′

end.

13. The method of claim 12, wherein the second chemical is an amine.

14. The method of claim 12, wherein the second chemical is methylamine.

15. The method of claim 12, wherein the second chemical is ethylenediamine.

16. The method of claim 12, wherein the second chemical is triethylamine.