Compound for sequencing by synthesis

Patent metadata

Field	Value
Publication number	US-2015140561-A1
Application number	US-201414542980-A
Country	US
Kind code	A1
Filing date	Nov 17, 2014
Priority date	Nov 20, 2013
Publication date	May 21, 2015
Grant date	—

Abstract

The present invention provides deoxynucleoside tri- or tetraphosphate comprising a 3′ nitrate and a detectable label covalently bound to the oxygen atom of an oxymethyl or oxyallyl or oxypropargyl substitution of a nucleobase. Such compounds provide new possibilities for future Sequencing by Synthesis technologies.

First claim

1 . A deoxynucleoside tri- or tetraphosphate comprising a 3′-nitrate and a detectable label covalently bound to the oxygen atom of an oxymethyl or oxyallyl or oxypropargyl substitution of a nucleobase.

2 . A deoxynucleoside tri- or tetraphosphate according to claim 1 , wherein said detectable label is a fluorescent label.

3 . A deoxynucleoside tri- or tetraphosphate according to claim 1 wherein said label is connected to the base via a cleavable linker.

4 . A kit comprising
a DNA polymerase, a pyrophosphatase, and at least one deoxynucleoside tri- or tetraphosphate according to claim 1 .

5 . A reaction mixture comprising
a nucleic acid template with a primer hybridized to said template, a DNA polymerase, a pyrophosphatase, and at least one deoxynucleoside tri- or tetraphosphate according to claim 1 .

6 . A method of performing a DNA synthesis reaction comprising the steps of
preparing a reaction mixture by means of providing a nucleic acid template with a primer hybridized to said template, a DNA polymerase, at least one deoxynucleoside tri- or tetraphosphate according to claim 1 , and subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction.

7 . A method according to claim 6 , wherein said DNA synthesis reaction is a sequencing reaction.

8 . A method for analyzing a DNA sequence comprising the steps of
a) providing a nucleic acid template with a primer hybridized to said template forming a primer/template hybridization complex, b) adding DNA polymerase, and at least one labeled deoxynucleoside tri- or tetraphosphate according to claim 1 , and a pyrophosphatase, c) subjecting said reaction mixture to conditions which enable a DNA polymerase catalyzed primer extension reaction d) determining, whether said compound has been incorporated into said primer/template hybridization complex.

9 . A method according to claim 8 , further comprising the steps of
e) removing said blocking group and optionally said label from said complex, and f) repeating steps b) to e) at least once.

10 . A method according to claim 9 , wherein a different labeled deoxynucleoside tri- or tetraphosphate compound is added during repeat of step b).

11 . A method according to claim 9 , wherein a mixture of 4 differently labeled deoxynucleoside tri- or tetraphosphate compounds representing A, G, C and T/U are used in step b).

12 . A method according to claim 9 , wherein step e) is performed by means of adding a sulfide or a mercaptane.

Assignees

• Roche Diagnostics Operations

Inventors

• Bergmann Frank
• Heindl Dieter
• Steger-Domandl Jessica

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title