ENZYMATIC NUCLEIC ACID SYNTHESIS: COMPOSITIONS AND METHODS FOR INHIBITING PYROPHOSPHOROLYSIS

• US 20160244822A1
• Filed: 12/21/2015
• Published: 08/25/2016
• Est. Priority Date: 12/01/2000
• Status: Active Grant

• 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
• Granted Patent: US 9,845,500 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 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 method for sequencing nucleic acids, comprising:

a) flowing labeled deoxynucleotide triphosphates onto template nucleic acids which are hybridized to a primer nucleic acid, where the template nucleic acids or primers are attached to a support, and wherein the labeled nucleotide triphosphates include a molecular tag bonded to the beta- or gamma-phosphate;

b) incorporating one of the labeled nucleotide triphosphates into at least one of the primers with a polymerase;

c) releasing a tagged pyrophosphate which includes the molecular tag bonded to the beta- or gamma-phosphate; and

d) detecting the released tagged pyrophosphates.

2. The method of claim 1, further comprising:

flowing in step (a) at least one 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 2, 3, 4 or 5.

3. The method of claim 1, wherein the support comprises a micro-fabricated chip.

4. The method of claim 1, wherein the template nucleic acids or the primers that are hybridized to the template nucleic acids are attached to the support in an ordered array.

5. The method of claim 1, wherein the flowing I step (a) includes a single type of labeled deoxynucleotide triphosphates selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxyuridine triphosphate.

6. The method of claim 1, wherein the released tagged pyrophosphates are detected in parallel.

7. The method of claim 1, wherein the molecular tag comprises aminonaphthalene-1-sulfonate (ANS).

8. The method of claim 1, wherein the molecular tag comprises an alkyl group, aryl group, alkaryl group, aralkyl group.

9. The method of claim 1, wherein the released tagged pyrophosphate comprise a nitrogen or sulfur atom that substitutes for any of the oxygen atoms