Terminal phosphate blocked nucleoside polyphosphates

• US 20040048300A1
• Filed: 08/29/2003
• Published: 03/11/2004
• Est. Priority Date: 08/29/2001
• Status: Active Grant

• Current Assignee: Bioelectric Medical Solutions, Inc., Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Inventors: Nelson, John, Sood, Anup, Kumar, Shiv, Fuller, Carl
• Granted Patent: US 7,256,019 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: 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: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The present invention describes terminal phosphate blocked nucleoside polyphosphates that are stable at high temperature and their use in nucleic acid amplification and analysis. Current invention further describes charge modified terminal phosphate blocked nucleoside polyphosphates for improved incorporation and direct loading of nucleic acid sequencing reactions onto separating media.

Claims

1. In a method for nucleic acid analysis, which includes a polymerase reaction of a nucleic acid template, a primer, a nucleic acid polymerase, and at least one nucleoside polyphosphate, the improvement comprising conducting said polymerase reaction in the presence of at least one terminally blocked nucleoside polyphosphates.

2. The method of claim 1, wherein said nucleic acid analysis is DNA sequencing.

3. The method of claim 2, wherein said terminally blocked nucleoside polyphosphate is a labeled terminator.

4. The method of claim 1, wherein said nucleic acid analysis is nucleic acid amplification.

5. The method of claim 4, wherein said amplification is done by polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA) or strand displacement amplification (SDA).

6. The method of claim 1, wherein said at least one terminally blocked nucleoside polyphosphate has additional negative charges.

7. The method of claim 1, wherein said at least one terminally blocked nucleoside polyphosphate is net positively charged.

8. The method of claim 1, wherein said at least one terminally blocked nucleoside polyphosphate is thermally stable.

9. The method of claim 1, wherein said terminally blocked nucleoside polyphosphates are represented by the formula

10. The method of claim 9, wherein at least one of L, B, S, X or Z is substituted with a detectable moiety.

11. The method of claim 9, wherein at least one of L, B, S, X or Z is substituted with a negatively charged moiety.

12. The method of claim 9, wherein at least one of L, B, S, X or Z is substituted with a moiety that impart a net positive charge.

13. The method of claim 9, wherein Z is modified to contain additional negative charges or one or more positive charges.

14. The method of claim 9, wherein L is labeled with a detectable moiety selected from the group consisting of radioisotopes, electrochemical tags, fluorescent tags, energy transfer (ET) labels, mass spectrometry tags, Raman tags, haptens, chemiluminescent groups, enzymes, chromophores, and any combinations thereof.

15. The method of claim 9, wherein B is selected from the group consisting of uracil, cytosine, guanine, adenine, thymine, 7-deazaguanine, 7-deazaadenine, hypoxanthine, 7-deazahypoxanthine and 2,6-diaminopurine or derivatives thereof.

16. The method of claim 9, wherein X is a polyphosphate chain of 3, 4 or 5 phosphate groups.

17. The method of claim 9 for sequencing, wherein S is selected from the group consisting of 2′

-deoxyribosyl, 3′

-deoxyribosyl, 2′

,3′

-dideoxyribosyl, 2′

,3′

-dideoxy-2′

,3′

-didehydroribosyl, 3′

-alkoxyribosyl, 3′

-azidoribosyl, 3′

-aminoribosyl, 3′

-fluororibosyl, 3′

-mercaptoribosyl, 3′

-alkylthioribosyl, 2′

-alkoxyribosyl, 2′

-azidoribosyl, 2′

-aminoribosyl, 2′

-fluororibosyl, 2′

-mercaptoribosyl, 2′

-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.

18. The method of claim 9 for nucleic acid amplification, wherein S is selected from the group consisting of ribosyl, 2′

-deoxyribosyl, 2′

-alkoxyribosyl, 2′

-azidoribosyl, 2′

-aminoribosyl, 2′

-fluororibosyl, 2′

-mercaptoribosyl, 2′

-alkylthioribosyl.

19. The method of claim 9, wherein said terminally blocked nucleoside polyphosphate is represented by the formula

20. A kit for DNA sequencing or nucleic acid amplification, comprising:

(a) at least one terminal phosphate blocked nucleoside polyphosphate; and

(b) a DNA polymerase.