Polymerization of nucleic acids using activation by polyphosphorolysis (APP) reactions

• US 9,476,093 B2
• Filed: 01/09/2015
• Issued: 10/25/2016
• Est. Priority Date: 12/13/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Xi, Lei, Kenney, Paul, Ma, Zhaochun, Prosen, Dennis, Hendricks, Stephen, Nagel, Roland
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/593,858
• Publication Number:
  US 20150191775A1
• Time in Patent Office: 655 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

This disclosure relates to methods of performing activation by polyphosphorolysis (APP) reactions using at least one of the polyphosphorylating agents triphosphate, polyphosphate, imidodiphosphate, thiodiphosphate (or μ-monothiopyrophosphate), and related compounds.

Claims

1. A method for amplifying a target nucleic acid by an activation by polyphosphorolysis (APP) reaction comprising the steps of:

(a) annealing to a template strand of the target nucleic acid a complementary activatable oligonucleotide P* having a non-extendable nucleotide at its 3′

terminus and having no mismatched nucleotides at or near its 3′

terminus, so that the terminal nucleotide is hybridized to the template strand when the oligonucleotide P* is annealed,

(b) polyphosphorolyzing the resulting duplex with;

1) one or more polyphosphorolyzing agents selected from the group consisting of a compound of the general formula I;

2. The method of claim 1 wherein the enzyme is a DNA polymerase.

3. The method of claim 2 wherein the enzyme is a thermostable DNA polymerase.

4. The method of claim 3 wherein the thermostable DNA polymerase is selected from the group consisting of thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1, RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATOR GAMMA.

5. The method of claim 1 wherein the extending and amplification steps are carried out using a polymerase chain reaction.

6. The method of claim 5 wherein the polymerase chain reaction is a digital polymerase chain reaction.

7. The method of claim 1 using one or more polyphosphorolyzing agents of Formula I:

8. The method of claim 1 using one or more polyphosphorolyzing agents of Formula II:

9. The method of claim 1 using one or more polyphosphorolyzing agents selected from the group consisting of:

10. The method of claim 9 using one or more polyphosphorolyzing agents selected from the group consisting of triphosphate, tetraphosphate, and imidodiphosphate.

11. The method of claim 1, wherein the polyphosphorolyzing further comprises using pyrophosphate in combination with the one or more polyphosphorolyzing agents.

12. The method of claim 1, further comprising detecting the polymerized nucleic acid.

13. The method of claim 1, wherein the one or more polyphosphorolyzing agents is present in the amplification reaction at a concentration of approximately 1-500 μ

M.

14. The method of claim 1, wherein the target nucleic acid is RNA.

15. The method of claim 14 wherein the enzyme or polymerase has reverse transcriptase activity.

16. The method of claim 14 wherein the enzyme is RQ1 or its mutant RQY.

17. The method of claim 14 used in a direct RNA sequencing reaction.