Enzymatic nucleic acid synthesis: methods for inhibiting pyrophosphorolysis during sequencing synthesis

• US 20070172869A1
• Filed: 12/29/2006
• Published: 07/26/2007
• Est. Priority Date: 12/01/2000
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Willson, Richard, Hardin, Susan, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Application Number:
  US11/648,723
• Publication Number:
  US 20070172869A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

Nucleotide triphosphate probes containing a molecular and/or atomic tag on a γ and/or β phosphate group and/or a base moiety having a detectable property are disclosed, and kits and method for using the tagged nucleotides in sequencing reactions and various assay. Also, phosphate and polyphosphate molecular fidelity altering agents are disclosed.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. A method of inhibiting or preventing pyrophosphorolysis during synthesis of a nucleic acid molecule, said method comprising:

combining a primer with a nucleic acid template under conditions sufficient to form a hybridized product; and

incubating the hybridized product with a polymerizing agent in the presence or absence of an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with alcohol group as acceptor, a nucleotidyltransferase, and a carboxy-lyase, under conditions sufficient to form a second nucleic acid molecule complementary to all or a portion of the nucleic acid template,

adding a nucleotide including an atomic and/or molecular tag on a β and

/or γ

-phosphate thereof to either or both steps to inhibit or prevent pyrophosphorolysis during synthesis of a nucleic acid molecule.

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. A primer extension reaction method comprising the steps of:

providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3′

termini of the first strand of said nucleic acid molecule and said second primer is complementary to a sequence at or near the 3′

termini of the second strand of said nucleic acid molecule;

hybridizing said first primer to said first strand and said second primer to said second strand in the presence of a polymerase, and optionally an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as an acceptor, a nucleotidyltransferase and a carboxy-lyase under conditions such that a third nucleic acid molecule complementary to said first strand and a fourth nucleic acid molecule complementary to said second strand are synthesized from nucleotides having a molecular and/or atomic tag bonded to or associated with a β

- and/or γ

-phosphate moiety of the nucleotide;

denaturing said first and third strand and said second and fourth strand; and

repeating steps (a) to (c) one or more times,

where the PP_i released during nucleotide polymerization includes the tag preventing deleterious effects that nascent PP_i cause without having to enzymatically degrade the released tagged PP_i and where the tagged nucleotides prevent inhibition of nucleic acid synthesis during amplification and prevent band drop out in sequencing reactions.

22. A method of sequencing a DNA molecule comprising:

combining a tagged primer with a first DNA molecule under conditions sufficient to form a hybridized product;

contacting said hybridized product with nucleotides having a molecular and/or atomic tag bonded to or associated with a β

- and/or γ

-phosphate moiety thereof, a DNA polymerase, optionally an enzyme selected from the group consisting of a pentosyltransferase, a phosphotransferase with an alcohol group as acceptor, a nucleotidyltransferase and a carboxy-lyase, and a terminator nucleotide to give a reaction mixture;

incubating the reaction mixture under conditions sufficient to synthesize a population of DNA molecules complementary to said first DNA molecule, wherein said synthesized DNA molecules are shorter in length than said first DNA molecule and wherein said synthesized DNA molecules comprise a terminator nucleotide at their 3′

termini; and

separating said synthesized DNA molecules by size so that at least a part of the nucleotide sequence of said first DNA molecule can be determined,

where the tagged nucleotides reduce band drop out and band spreading.

23. The method of claim 22, wherein the tag on the nucleotide is not fluorescent improving traditional fluorescent sequencing due to a decrease in background and/or a reduction if band spreading.

24. The method of claim 22, wherein the β

- and/or γ

-phosphate modified deoxynucleotide and/or dideoxynucleotides improve polymerase accuracy or fidelity.

25. The method of claim 22, wherein the β

- and/or γ

-phosphate modified nucleotides improve the production of identical (or substantially identical) DNA polymers instead of the production of a population of molecules that result from more frequent mis-incorporated nucleotides.

26. A method of identifying a base at a target position in a sample DNA sequence comprising the steps of:

providing an extension primer, which hybridizes to the sample DNA immediately adjacent to a target position and

subjecting the sample DNA and extension primer to a polymerase reaction in the presence of a first deoxynucleotide type having a molecular and/or atomic tag on a β

- and/or γ

-phosphate thereof or a first dideoxynucleotide type having a molecular and/or atomic tag on a β

- and/or γ

-phosphate thereof, where the tagged deoxynucleotide type or tagged dideoxynucleotide type will only become incorporated and release tagged pyrophosphate (tPPi) if it is complementary to the target position,

detecting a detectable property of an incorporated tagged deoxynucleotide and/or release tPPi or an incorporated tagged dideoxynucleotide and/or released tPPi, and

identifying the incorporated deoxynucleotide or dideoxynucleotide type.

27. The method of claim 26, further comprising the step of:

repeating the subjecting, detecting and identifying step until the deoxynucleotide or dideoxynucleotide type complementary to the target site is identified.

28. The method of claim 26, wherein the detecting step comprises detecting the released tPPi.