Method for isothermal DNA amplification starting from an RNA template

Patent metadata

Field	Value
Publication number	US-9777319-B2
Application number	US-201213538955-A
Country	US
Kind code	B2
Filing date	Jun 29, 2012
Priority date	Jun 29, 2012
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

Abstract

A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs); and amplifying the cDNA to form an amplified product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3′ to an inosine residue of the primer. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided.

First claim

The invention claimed is:

1. An isothermal method for generating an amplified cDNA from an RNA template in a single tube, the method comprising:
(a) providing the RNA template;
(b) reverse transcribing the RNA template under an isothermal condition to generate a cDNA:RNA heteroduplex by incubating the RNA template with a reaction mixture comprising at least one primer capable of hybridizing to the RNA template, a reverse transcriptase with reduced RNAse H activity, and deoxynucleoside triphosphates (dNTPs); and
(c) amplifying the cDNA of the cDNA:RNA heteroduplex under the same isothermal condition to form the amplified cDNA by incubating the reaction mixture of step (b) with an amplification reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer capable of hybridizing to the cDNA strand of the cDNA:RNA heteroduplex, an endonuclease capable of nicking a double stranded DNA 3′ to an inosine residue, deoxynucleoside triphosphates (dNTPs), glycerol, formamide, and ethylene glycol,
wherein the amplification reaction mixture has a low salt concentration to allow the at least one inosine-containing primer to hybridize with the cDNA:RNA heteroduplex without heat-denaturation of the cDNA:RNA heteroduplex, and
wherein the steps (a)-(c) are performed in the single tube under the said isothermal condition at a temperature in a range from about 40° C. to about 50° C.

2. The method of claim 1 , wherein the at least one primer capable of hybridizing to the RNA template comprises an oligo (Dt) primer, a Locked Nucleic Acid (LNA), a Peptide Nucleic Acid (PNA) or combinations thereof.

3. The method of claim 1 , wherein the amplified cDNA is complementary to at least a portion of the cDNA of the cDNA:RNA heteroduplex.

4. The method of claim 1 , wherein the amplification reaction mixture of step (c) comprises multiple forward and reverse inosine-containing primers capable of hybridizing to the cDNA strand of the cDNA:RNA heteroduplex.

5. The method of claim 1 , wherein the reverse transcriptase with reduced RNAse H activity is selected from one or more of a mutated form of MMLV reverse transcriptase, a mutated form of AMY reverse transcriptase, a mutated form of HIV reverse transcriptase, or conservative variants thereof.

6. The method of claim 1 , wherein at least one inosine nucleotide of the at least one inosine-containing primer is positioned at least 4 nucleotides from the 5′ end of the at least one inosine-containing primer.

7. The method of claim 1 , wherein the at least one inosine-containing primer comprises a phosphorothioate linkage.

8. The method of claim 1 , wherein the amplification reaction mixture comprises at least one extender template.

9. The method of claim 1 , wherein the at least one inosine-containing primer has a sequence length ranging between 5 to 100, 5 to 30, or 5 to 20 nucleotides.

10. The method of claim 1 , wherein the at least one strand displacement DNA polymerase is an exonuclease deficient polymerase.

11. The method of claim 1 , wherein the at least one strand displacement DNA polymerase is selected from a group consisting of Bst DNA polymerase, an exonuclease deficient T7 DNA polymerase, exo (−) Klenow, delta Tts DNA polymerase, BCA DNA polymerase, Phi 29 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase and combinations thereof.

12. The method of claim 1 , wherein the endonuclease is an endonuclease V.

13. The method of claim 12 , wherein the endonuclease V is selected from a protein with a sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27 or conservative variants thereof.

14. The method of claim 1 , wherein a final concentration of the dNTPs comprising dTTP, dCTP, dATP and dGTP in the reaction mixture is in a range of about 10 μM to 10,000 μM.

15. The method of claim 1 , wherein the amplification reaction mixture further comprises a surfactant.

16. The method of claim 1 , wherein the amplification reaction mixture further comprises a divalent cation selected from Mn 2+ , Mg 2+ or a combination thereof.

17. The method of claim 1 , wherein the amplification reaction mixture further comprises a reducing agent selected from dithiothreitol (DTT), 2-mercaptoethanol (β-ME), 2-mercaptoethylamine (MEA), or Tris(carboxyethyl) phosphine (TCEP).

18. The method of claim 1 , wherein the amplification reaction mixture further comprises at least one single stranded DNA binding protein (SSB).

19. The method of claim 18 , wherein the at least one single stranded DNA binding protein is selected from a group consisting of E. coli SSB, T4 gene 32 protein, T7 gene 15 protein, Ncp7, recA and combinations thereof.

20. The method of claim 1 , wherein the RNA template is isolated from eukaryotic origin, prokaryotic origin, viral origin, bacteriophage origin, or synthetic origin.

21. The method of claim 1 , wherein the reverse transcriptase with reduced RNAse H activity is a mutated form of MMLV reverse transcriptase.

22. The method of claim 1 , further comprising quantifying the RNA template by measuring the rate of amplification of the cDNA.

Assignees

• Nelson John Richard
• Duthie Robert Scott
• Grossmann Gregory Andrew
• Heller Ryan Charles
• Gen Electric

Inventors

• Nelson John Richard
• Duthie Robert Scott
• Grossmann Gregory Andrew
• Heller Ryan Charles

Classifications

• C12Q1/6865 Primary

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• C12Q2545/114

  involving a quantitation step · CPC title

• C12Q2525/117

  incorporating modified base · CPC title