Pyrosequencing methods and related compositions

• US 9,169,510 B2
• Filed: 06/20/2006
• Issued: 10/27/2015
• Est. Priority Date: 06/21/2005
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Ju, Jingyue, Wu, Jian, Kim, Dae H.
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US11/922,385
• Publication Number:
  US 20090325154A1
• Time in Patent Office: 3,416 Days
• Field of Search: 435/6.1, 435/283.1, 435/287.2, 536/23.1, 422/430
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2334/20 Coumarin derivatives
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

This invention provides methods for pyrosequencing and compositions comprising 3′-O— modified deoxynucleoside triphosphates.

Claims

1. A method for determining the nucleotide sequence of a single-stranded DNA comprising performing the following steps:

(a) immobilizing the single-stranded DNA on a solid substrate via an azido linkage, an alkynyl linkage, or a 1,2,3-triazole linkage;

(b) contacting the DNA under DNA polymerization-permitting conditions with (i) a 3′

-O-blocked dNTP selected from the group consisting of 3′

-O-blocked dATP, 3′

-O-blocked dCTP, 3′

-O-blocked dGTP, and 3′

-O-blocked dTTP, wherein each 3′

-O-blocked dNTP comprises an unmodified triphosphate group, and wherein each 3′

-O-blocked dNTP comprises an unlabeled base, and (ii) 9°

N DNA polymerase (exo-) A4851/Y409V, wherein the moiety blocking the 3′

-O atom of the dNTP is an allyl moiety or a methoxymethyl moiety;

(c) (i) determining whether pyrophosphate is generated as a result of step (b), whereby (1) pyrophosphate generation indicates that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′

-O-blocked dNTP used in part (i) of step (b), and (2) the absence of pyrophosphate generation indicates that the identity of such nucleic acid residue is not that which is complementary to such 3′

-O-blocked dNTP;

wherein determining whether pyrophosphate is generated is performed by detecting dissociation of a coumarin-derived indicator from a complex between the indicator and a bis-Zn²⁺-dipicolylamine coordination compound, wherein the coumarin-derived indicator has the following structure;

2. The method of claim 1, wherein the moiety is an allyl moiety.

3. The method of claim 1, wherein the moiety blocking the 3′

-O atom of the dNTP is an allyl moiety and removing it is performed using Na₂PdCl₄ and 3,3′

,3″

-phosphinidynetris(benzenesulfonic acid), trisodium salt (TPPTS).

4. The method of claim 1, wherein the solid substrate is in the form of a chip, a bead, a well, a capillary tube, or a slide.

5. The method of claim 1, wherein the solid substrate is gold, quartz, silica, or plastic.

6. The method of claim 1, wherein the solid substrate is porous.