Methods and compositions for incorporating nucleotides

• US 9,017,973 B2
• Filed: 11/28/2011
• Issued: 04/28/2015
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: IsoPlexis Corporation (Bruker Corporation)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Gordon, Steven, Olejnik, Jerzy
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/305,415
• Publication Number:
  US 20130137091A1
• Time in Patent Office: 1,247 Days
• Field of Search: 435/6.1, 435/91.1, 435/91.2, 435/91.5, 536/24.3, 536/26.6
• US Class Current: 435/91.5
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/058 Flat flow cell
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 2021/6471 Special filters, filter wheel
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/645 Specially adapted constructive features of fluorimeters
• CPC Class Codes: G01N 2201/062 LED's
• CPC Class Codes: G01N 2201/0627 : Use of several LED's for spectral resolution
• CPC Class Codes: G01N 2201/12 : Circuits of general importance; Signal processing
• CPC Class Codes: G16B 40/10 : Signal processing, e.g. from mass spectrometry [MS] or from PCR

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

Claims

1. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a solution comprising a first plurality of nucleotide analogues wherein each nucleotide analogue is labeled with a unique label and contains a removable chemical moiety capping the 3′

-OH group, and a second plurality of nucleotide analogues wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′

-OH group; and

iii) polymerase;

b) introducing said solution into said reaction chamber under conditions wherein a nucleotide analogue of said first plurality of nucleotide analogues is incorporated by said polymerase; and

c) detecting the label of the incorporated nucleotide analogue.

2. The method of claim 1, wherein said polymerase is in said solution prior to step b).

3. The method of claim 1, wherein said first nucleotide is incorporated at step b) into a plurality of primers hybridized to said plurality of nucleic acid template molecules.

4. The method of claim 1, wherein each of said plurality of nucleic acid template molecules comprises a self-priming hairpin, and said first nucleotide is incorporated at step b) into said template.

5. The method of claim 1, wherein the second plurality of nucleotide analogues is present in said solution at a higher concentration than said first plurality of nucleotide analogues.

6. The method of claim 1, wherein said second plurality of nucleotide analogues is present at a concentration between 1 uM and 100 uM.

7. The method of claim 1, wherein said first plurality of nucleotide analogues is present at a concentration between 1 nM and 1 uM.

8. The method of claim 1, wherein said first plurality of nucleotide analogues comprises four different nucleotide analogues.

9. The method of claim 8, wherein said second plurality of nucleotide analogues comprises four different nucleotide analogues.

10. The method of claim 8, wherein each of said four different nucleotide analogues comprises a unique label, said label selected from the group consisting of BODIPY, Rhodamine, Carboxyrhodamine, and Cyanine.

11. The method of claim 1, wherein said removable chemical moiety comprises a disulfide bond.

12. The method of claim 1, wherein said removable chemical moiety comprises an azido group.

13. The method of claim 1, wherein said cleavable linker comprises a disulfide bond.

14. The method of claim 1, wherein said removable chemical moiety comprises an azido group and said cleavable linker comprises a disulfide bond.

15. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a solution comprising a first plurality of four different nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached via a cleavable linker and contains a removable chemical moiety capping the 3′

-OH group, and a second plurality of nucleotide analogues wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′

-OH group; and

iii) polymerase;

b) introducing said solution into said reaction chamber under conditions wherein a nucleotide analogue of said first plurality of nucleotide analogues is incorporated by said polymerase; and

c) detecting the label of the incorporated nucleotide analogue.

16. The method of claim 15, wherein said polymerase is in said solution prior to step b).

17. The method of claim 15, wherein said first nucleotide is incorporated at step b) into a plurality of primers hybridized to said plurality of nucleic acid template molecules.

18. A method of incorporating labeled nucleotides into nucleic acid, comprising:

a) providing i) a reaction chamber comprising plurality of nucleic acid template molecules bound to a solid support, ii) a first solution comprising a first plurality of four different nucleotide analogues wherein each nucleotide analogue is labeled with a unique label attached via a cleavable linker and contains a removable chemical moiety capping the 3′

-OH group, and a second plurality of nucleotide analogues wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′

-OH group, iii) a second solution lacking labeled nucleotide analogues and comprising a plurality of nucleotide analogues wherein each nucleotide analogue is unlabeled and contains a removable chemical moiety capping the 3′

-OH group, and iv) polymerase;

b) introducing said first solution into said reaction chamber under conditions wherein a nucleotide analogue of said first plurality of nucleotide analogues is chamber; and

d) detecting the label of the incorporated nucleotide analogue.

19. The method of claim 18, wherein said polymerase is in said solution prior to step b).

20. The method of claim 18, wherein said first nucleotide is incorporated at step b) into a plurality of primers hybridized to said plurality of nucleic acid template molecules.

21. The method of claim 18, further comprising washing before step d).

22. The method of claim 18, further comprising removing the label of the incorporated analogue.

23. The method of claim 22, further comprising repeating steps b), c) and d).