US-12618109-B2 - Orthogonal deblocking of nucleotides

Abstract

A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.

Inventors

• Eliane H. Trepagnier
• Tarun Khurana

Assignees

• ILLUMINA, INC.

Dates

Publication Date

20260505

Application Date

20211013

Claims (20)

1. 1 . A nucleic acid array, comprising a plurality of sites on a solid support, wherein each site comprises a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence that is different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, a first reversible blocking moiety being attached to the first primer, wherein a second primer is bound to the second nucleic acid template, a second reversible blocking moiety being attached to the second primer, and wherein the first reversible blocking moiety is different from the second reversible blocking moiety, and wherein the first primer and second primer comprise the first reversible blocking moiety and second reversible blocking moiety, respectively, simultaneously.
2. 2 . The nucleic acid array of claim 1 , wherein each site occupies an area on the solid support that is 100 μm 2 or less.
3. 3 . The nucleic acid array of claim 1 , wherein the plurality of sites has a pitch of 10 μm or less.
4. 4 . The nucleic acid array of claim 1 , wherein the plurality of sites comprises at least 1×10 6 sites.
5. 5 . The nucleic acid array of claim 4 , wherein each of the sites comprises a nucleic acid sequence that is unique compared to the nucleic acid sequences at the other sites in the plurality.
6. 6 . The nucleic acid array of claim 1 , wherein the first reversible blocking moiety is attached to the 3′ nucleotide of the first primer.
7. 7 . The nucleic acid array of claim 6 , wherein the 3′ nucleotide of the first primer is attached to a first optical label.
8. 8 . The nucleic acid array of claim 1 , wherein the second reversible blocking moiety is attached to the 3′ nucleotide of the second primer.
9. 9 . The nucleic acid array of claim 8 , wherein the 3′ nucleotide of the second primer is attached to a second optical label, wherein the second optical label is optically distinguishable from a first optical label.
10. 10 . The nucleic acid array of claim 9 , wherein the first and second optical labels comprise fluorophores.
11. 11 . The nucleic acid array of claim 1 , wherein the first nucleic acid template and the second nucleic acid template comprise DNA.
12. 12 . The nucleic acid array of claim 1 , wherein a single nucleic acid molecule contains the first nucleic acid template and the second nucleic acid template.
13. 13 . The nucleic acid array of claim 1 , wherein the first nucleic acid template and the second nucleic acid template are on different nucleic acid molecules.
14. 14 . The nucleic acid array of claim 1 , wherein the sites comprise multiple amplicons of the first nucleic acid template and multiple amplicons of the second nucleic acid template.
15. 15 . The nucleic acid array of claim 1 , wherein the first primer comprises a first universal primer sequence and the first nucleic acid template at each site in the plurality of sites comprises a first universal primer binding sequence that is complementary to the first universal primer sequence.
16. 16 . The nucleic acid array of claim 15 , wherein the second primer comprises a second universal primer sequence and the second nucleic acid template at each site in the plurality of sites comprises a second universal primer binding sequence that is complementary to the second universal primer sequence, wherein the first universal primer binding sequence is different from the second universal primer binding sequence.
17. 17 . The nucleic acid array of claim 1 , wherein a first polymerase is bound to the first primer and the first nucleic acid template.
18. 18 . The nucleic acid array of claim 17 , wherein a second polymerase is bound to the second primer and the second nucleic acid template, and wherein the first polymerase and second polymerase are the same species of polymerase.
19. 19 . The nucleic acid array of claim 1 , wherein the first reversible blocking moiety comprises azidomethyl and wherein the second reversible blocking moiety comprises tert-butoxy-ethoxy.
20. 20 . A detection apparatus, comprising the nucleic acid array of claim 1 , and a detector positioned to detect the plurality of sites.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisional of U.S. application Ser. No. 15/739,587, filed Dec. 22, 2017 (now U.S. Pat. No. 11,155,864), which application is a U.S. National Stage Application filed under 35 U.S.C. § 371 claiming priority to International Application No. PCT/US2016/041568, filed Jul. 8, 2016, which application claims priority to U.S. Provisional Application Ser. No. 62/198,947, filed Jul. 30, 2015, the disclosures of which are incorporated herein by reference in their entirety. BACKGROUND This disclosure relates generally to nucleic acid analysis, and more specifically to nucleic acid sequencing. Currently available commercial platforms for sequencing DNA are relatively costly. These platforms use a ‘sequencing-by-synthesis’ approach, so called because DNA polymers are synthesized while detecting the addition of each monomer (i.e. nucleotide) to the growing polymer structure. Because a template DNA strand strictly directs synthesis of a new DNA polymer, one can infer the sequence of the template DNA from the series of nucleotide monomers that were added to the growing strand during the synthesis. The ability to detect monomer additions is facilitated by specially engineered variants of the biochemical components that normally carry out DNA synthesis in biological systems. These engineered components are relatively expensive to make and are consumed in relatively large amounts during sequencing-by-synthesis. Furthermore, monitoring the reaction uses relatively expensive hardware such as lasers, detection optics and complex fluid delivery systems. The most successful commercial platforms to date also require expensive reagents and hardware to amplify the DNA templates before sequencing-by-synthesis can even begin. The complexity and expense of these platforms has hindered their use in some clinical and research contexts where there is a clear need for the technology. Thus, there exists a need for improvements to sequencing-by-synthesis platforms to make them more cost effective, rapid and convenient. The present disclosure addresses this need and provides other advantages as well. BRIEF SUMMARY The present disclosure provides a method for identifying nucleic acid templates. The method can include steps of (a) providing an array of sites, wherein each site comprises a mixture of at least two different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties. Optionally, the method can further include (e) repeating (b) through (d) to determine the sequence of different nucleotide analogs added to each of the different extension products at each of the sites. Also provided is a method for sequencing nucleic acid templates that can include the steps of (a) providing an array of sites, wherein each site includes a first nucleic acid template and a second nucleic acid template, wherein the first nucleic acid template has a sequence that is different from the sequence of the second nucleic acid template, wherein a first primer is bound to the first nucleic acid template, and wherein a second primer is bound to the second nucleic acid template, a reversible blocking moiety being attached to the second primer; (b) extending the first primer by addition of a first nucleotide analog that is attached to a reversible blocking moiety, wherein the reversible blocking moiety that is attached to the first nucleotide is different from the reversible blocking moiety that is attached to the second primer; (c) selectively removing the reversible blocking moiety that is attached to the second primer while retaining the reversible blocking moiety that is attached to the nucleotide analog that is added to the first primer; (d) extending the second primer by addition of a second nucleotide analog that is attached to a reversible blocking moiety, wherein the reversible blocking moiety that is attached to the first nucleotide analog is different from the reversible blocking moiety that is attached to the second nucleotide analog; and (e) detecting the nucleotide analog that is added to the first primer and the nucleotide analog that is added to the second primer, at each of the sites, thereby determining the different sequences of the first template and the second template at each of the sites. Optionally, the method can further include steps of (f