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

• US 8,932,813 B2
• Filed: 12/13/2011
• Issued: 01/13/2015
• Est. Priority Date: 12/13/2010
• Status: Expired due to Fees

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kenney, Paul, Ma, Zhaochun, Prosen, Dennis, Hendricks, Stephen, Nagel, Roland, Xi, Lei (Larry)
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/324,676
• Publication Number:
  US 20120196329A1
• Time in Patent Office: 1,127 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 435/6.1
• 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 producing an unblocked oligonucleotide from a blocked oligonucleotide comprising a non-extendable 3′

end removable by polyphosphorolysis, the method comprising

contacting the blocked oligonucleotide hybridized to a target nucleic acid with one or more polyphosphorolyzing agents selected from the group consisting of;

a compound of the general formula I;

2. The method of claim 1, further comprising extending the unblocked oligonucleotide to produce a desired nucleic acid strand.

3. The method of claim 2, further comprising amplifying the desired nucleic acid strand.

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

5. The method of claim 4 wherein the enzyme is a thermostable DNA polymerase.

6. The method of claim 5 wherein the thermostable DNA polymerase is selected from the group consisting of to thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1, RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATOR GAMMA.

7. The method of claim 3 wherein the extending and amplification steps are carried out using a polymerase chain reaction.

8. The method of claim 2 for polymerizing a target nucleic acid, the method comprising performing polymerization using an activation by polyphosphorolysis (APP) reaction using one or more polyphosphorolyzing agents of Formula I:

9. The method of claim 2 for polymerizing a target nucleic acid, the method comprising performing polymerization using an activation by polyphosphorolysis (APP) reaction using one or more polyphosphorolyzing agents of Formula II:

10. The method of claim 2 for polymerizing a target nucleic acid, the method comprising performing polymerization using an activation by polyphosphorolysis (APP) reaction using one or more polyphosphorolyzing agents selected from the group consisting of:

11. The method of claim 8 further comprising amplifying the nucleic acid molecule thus polymerized.

12. The method of claim 11, wherein the enzyme is a DNA polymerase.

13. The method of claim 12, wherein the enzyme is a thermostable DNA polymerase.

14. The method of claim 13, wherein the thermostable DNA polymerase is selected from the group consisting of to thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1, RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATOR GAMMA.

16. The method of claim 2, for polymerizing a target nucleic acid comprising performing polymerization using an activation by polyphosphorolysis (APP) reaction using one or more polyphosphorolyzing agents.

17. The method of claim 16, further comprising amplifying the nucleic acid molecule thus polymerized.

18. The method of claim 17, wherein the enzyme is a DNA polymerase.

19. The method of claim 17, wherein the enzyme is a thermostable DNA polymerase.

20. The method of claim 19, wherein the thermostable DNA polymerase is selected from the group consisting of to thermostable Tfl, Taq, AMPLITAQFS, THERMOSEQUENASE, RQ1, RQY, THERMINATOR I, THERMINATOR II, THERMINATOR III, and THERMINATOR GAMMA.

21. The method of claim 20 wherein the extending and amplification steps are carried out using a polymerase chain reaction.

22. The method of claim 3 wherein the reaction comprises the steps of:

(a) annealing to a nucleic acid a first oligonucleotide P* which has a non-extendable 3′

end, wherein the 3′

non-extendable terminus of the first oligonucleotide P* is removable by polyphosphorolysis;

(b) removing the 3′

non-extendable terminus of the first oligonucleotide P* annealed to the nucleic acid by polyphosphorolysis; and

,

(c) extending the unblocked first oligonucleotide using a nucleic acid polymerase; and

(d) repeating the steps (a)-(c) as necessary to amplify the target nucleic acid.

23. The method of claim 17, further comprising detecting the polymerized nucleic acid.

24. 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.

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

26. The method of claim 25 wherein the enzyme or polymerase has reverse transcriptase activity.

27. The method of claim 25 wherein the enzyme is RQ1 or its mutant RQY.

28. The method of claim 25 used in a direct RNA sequencing reaction.

15. The method of 11, wherein the extending and amplification steps are carried out using a polymerase chain reaction.