COMPOUND FOR SEQUENCING BY SYNTHESIS

• US 20150140561A1
• Filed: 11/17/2014
• Published: 05/21/2015
• Est. Priority Date: 11/20/2013
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Bergmann, Frank, Heindl, Dieter, Steger-Domandl, Jessica
• Granted Patent: US 9,410,197 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

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.

Claims

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.