Compound for sequencing by synthesis
First Claim
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1. A method for analyzing a DNA sequence comprising the steps ofa) 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 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, 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,e) simultaneously removing the 3′
-nitrate group and optionally said label from said complex by means of adding a sulfide or a mercaptane, andf) repeating steps b) to e) at least once.
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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.
7 Citations
3 Claims
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1. 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 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, 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, e) simultaneously removing the 3′
-nitrate group and optionally said label from said complex by means of adding a sulfide or a mercaptane, andf) repeating steps b) to e) at least once. - View Dependent Claims (2, 3)
- -nitrate and a detectable label covalently bound to the oxygen atom of an oxymethyl or oxyallyl or oxypropargyl substitution of a nucleobase, and a pyrophosphatase,
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Current AssigneeRoche Diagnostics Operations Incorporated (Roche Holding AG)
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Original AssigneeRoche Molecular Systems (Roche Holding AG)
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InventorsSteger-Domandl, Jessica, Bergmann, Frank, Heindl, Dieter
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Primary Examiner(s)Riley, Jezia
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Application NumberUS14/542,980Publication NumberTime in Patent Office631 DaysField of Search536/4.1, 536/23.1, 435/6.1, 435/91.1US Class Current1/1CPC Class CodesC07H 19/10 with the saccharide radical...C07H 19/20 with the saccharide radical...C12Q 1/6869 Methods for sequencingC12Q 2525/186 incorporating a non-extenda...
