Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis

• US 8,648,179 B2
• Filed: 10/04/2012
• Issued: 02/11/2014
• Est. Priority Date: 12/01/2000
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hardin, Susan H., Gao, Xiaolian, Briggs, James, Willson, Richard, Tu, Shiao-Chun
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/644,469
• Publication Number:
  US 20130102050A1
• Time in Patent Office: 495 Days
• Field of Search: 536/4.1, 536/23.1, 435/6.1, 435/91.1
• US Class Current: 536/4.1
• 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 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. A composition comprising a purified polymerase, a nucleic acid template molecule, and at least one nucleotide compound selected from the group consisting of compounds of the following general formulas:

Z—

OPO₂—

(OP(EE′

)O)_n—

PO₂O —

Z′

 

 

(e)

Z—

OPO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(f)

Z—

PO₂—

(OP(EE′

)O)_nPO₂O—

Z′

 

 

(g)

Z—

PO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(h)

where Z and Z′

are each independently a hydrogen atom or a thermally stable substituent, E and E′

are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least two.

5. The composition of claim 1 , wherein n is 2.

6. The composition of claim 1 , wherein n is at least 3.

7. The composition of claim 1 , wherein n is at least 4.

8. The composition of claim 1 , wherein n is at least 5.

2. A method for performing a nucleotide incorporation, comprising:

forming a nucleotide polymerization reaction mixture including a polymerase, a template nucleic acid molecule, and a nucleotide analog selected from the group consisting of nucleotide compounds having the following general formulas;

Z—

OPO₂—

(OP(EE′

)O)_n—

PO₂O —

Z′

 

 

(e)

Z—

OPO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(f)

Z—

PO₂—

(OP(EE′

)O)_nPO₂O—

Z′

 

 

(g)

and

Z—

PO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(h)

where Z and Z′

are each independently a hydrogen atom or a thermally stable substituent, E and E′

are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least 2; and

incorporating the nucleotide analog into a nascent nucleic acid molecule in a template-dependent manner using the polymerase.

3. A method for nucleotide incorporation, comprising:

forming a sequencing reaction mixture including a template nucleic acid molecule, a polymerase and at least one nucleotide compound having a formula selected from the group consisting of;

Z—

OPO₂—

(OP(EE′

)O)_n—

PO₂O —

Z′

 

 

(e)

Z—

OPO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(f)

Z—

PO₂—

(OP(EE′

)O)_nPO₂O—

Z′

 

 

(g)

and

Z—

PO₂—

(OP(EE′

)O)_nPO₂—

Z′

 

 

(h)

where Z and Z′

are each independently a hydrogen atom or a thermally stable substituent, E and E′

are each independently an oxygen atom or a thermally stable substituent, and n is an integer having a value of at least 2;

incorporating one or more nucleotide compounds of the sequencing reaction mixture into a nascent nucleic acid molecule in a template-dependent manner using the polymerase;

detecting a signal that indicates the incorporation of at least one of the nucleotide compounds into the nascent nucleic acid molecule by the polymerase.

4. The method of claim 3, further including identifying at least one of the incorporated nucleotide compounds, thereby obtaining sequence information from the template nucleic acid molecule.

9. The method of claim 3, wherein n is 2.

10. The method of claim 3, wherein n is at least 3.

11. The method of claim 3, wherein n is at least 4.

12. The method of claim 3, wherein n is at least 5.

13. A method for performing a nucleotide incorporation, comprising:

contacting a polymerase with a template nucleic acid molecule and a nucleotide including a polyphosphate chain including at least four phosphate groups, and incorporating the nucleotide into a nascent nucleic acid molecule in a template-dependent manner using the polymerase.

14. The method of claim 13, further including identifying the nucleotide that is incorporated into the nascent nucleic acid molecule.

15. The method of claim 13, further including:

detecting a signal indicating the template-dependent incorporation of the nucleotide into the nascent nucleic acid molecule by the polymerase.

16. The method of claim 15, wherein the nucleotide includes an optically detectable label that generates an optically detectable emission indicating the template-dependent incorporation.

17. The method of claim 16, wherein detecting the signal further includes detecting a change in the intensity, the frequency or the duration of the optically detectable emission.

18. The method of claim 16, wherein the polymerase tag includes a fluorescent resonance energy transfer (FRET) donor and the label of the nucleotide includes a FRET acceptor, and wherein the incorporating further includes performing energy transfer between the FRET donor and the FRET acceptor.

19. The method of claim 13, wherein the polyphosphate chain includes at least five phosphate groups.

20. The method of claim 17, further including identifying the nucleotide that is incorporated by analyzing the signal.

21. The method of claim 20, wherein the identifying the nucleotide includes determining the base identity of the nucleotide.