Additives to improve sequencing by synthesis performance

• US 10,519,498 B2
• Filed: 02/08/2017
• Issued: 12/31/2019
• Est. Priority Date: 02/11/2016
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Inventors: DiMeo, James J.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/427,718
• Publication Number:
  US 20170233805A1
• Time in Patent Office: 1,056 Days
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• 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 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The invention relates to methods, compositions, devices, systems and kits are described including, without limitation, reagents and mixtures, for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods.

Claims

1. A method of incorporating nucleotides, comprising:

a) providing i) a plurality of nucleic acid primers and template molecules, ii) an extend reagent comprising polymerase and a plurality of nucleotide analogues, and iii) a wash reagent comprising methylenediphosphonic acid;

b) hybridizing at least a portion of said primers to at least a portion of said template molecules so as to create hybridized primers;

c) exposing said hybridized primers to said extend reagent under conditions such that a first nucleotide analogue is incorporated into at least a portion of said hybridized primers so as to create extended primers comprising an incorporated nucleotide analogue;

d) washing said extended primers with said wash reagent.

2. The method of claim 1, wherein said incorporated nucleotide analogue comprises a label attached through a cleavable disulfide linker to the base.

3. The method of claim 2, wherein said label is fluorescent.

4. The method of claim 2, wherein said extend reagent further comprises cystamine.

5. The method of claim 2, further comprising:

e) detecting said label of a first labeled nucleotide analogue.

6. The method of claim 5, wherein said detecting of step e) is performed in the presence of methylenediphosphonic acid.

7. The method of claim 1, wherein the concentration of said methylenediphosphonic acid is between 0.1 mM and 4 mM.

8. The method of claim 1, wherein the concentration of said methylenediphosphonic acid is between 0.25 mM and 1 mM.

9. The method of claim 1, wherein the concentration of said methylenediphosphonic acid is between 0.35 mM and 0.65 mM.