ENZYMATIC NUCLEIC ACID SYNTHESIS: COMPOSITIONS AND METHODS FOR INHIBITING PYROPHOSPHOROLYSIS

• US 20140127781A1
• Filed: 12/20/2013
• Published: 05/08/2014
• 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 9,243,284 B2
• Time in Patent Office: Days
• US Class Current: 435/194
• 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 composition comprising a modified nucleotide including a molecular and/or atomic tag, a nucleotide polymerizing agent, a primer, a template and a pyrophosphorolysis inhibitor, where the modified nucleotide alters base incorporation fidelity of the nucleotide polymerizing agent relative to a base incorporation fidelity of the agent in the absence of the modified nucleotide and where the pryophosphorolysis inhibitor reduces pryophosphorolysis.

2. The composition of claim 1, wherein the modified nucleotide comprises a β

and/or γ

phosphate modified nucleotide.

3. The composition of claim 1, wherein the modified nucleotide comprises a β

phosphate modified nucleotide.

4. The composition of claim 1, wherein the modified nucleotide comprises a γ

phosphate modified nucleotide.

5. The composition of claim 4, wherein the tag comprises a molecule.

6. The composition of claim 5, wherein the tag is ANS.

7-58. -58. (canceled)