BI-DIRECTIONAL SEQUENCING COMPOSITIONS AND METHODS

• US 20150353994A1
• Filed: 08/21/2015
• Published: 12/10/2015
• Est. Priority Date: 07/07/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: MYERS, Jason, CHEN, Zhoutao, NIKIFOROV, Theo, DRESSMAN, Devin
• Granted Patent: US 9,587,269 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

In some embodiments, methods for obtaining sequence information from a nucleic acid template linked to a support include hybridizing a first primer to a template strand linked to a support, sequencing a portion of the nucleic acid template, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid template, In some embodiments, the method further includes introducing a nick into a portion of the template strand that is hybridized to the extended first primer product, degrading a portion of the template strand from the nick using a degrading agent, where a portion of the extended first primer remains hybridized to an undegraded portion of the template strand, and sequencing at least some of the single-stranded portion of the extended first primer by synthesis.

Claims

1. A method for generating single-stranded nucleic acids, comprising:

a) hybridizing a first primer to a nucleic acid molecule;

b) extending the first primer via polymerization under polymerization conditions, thereby forming an extended first primer product that is complementary to a portion of the nucleic acid molecule;

c) introducing a nick into a portion of the nucleic acid molecule that is hybridized to the extended first primer product, where the nick includes a free 5′

end and a free 3′

end in the nucleic acid molecule; and

d) degrading the nucleic acid molecule from the free 5′

end of the nick using a degrading agent, thereby generating a single-stranded extended primer product which remains hybridized to an undegraded portion of the nucleic acid molecule.

2. The method of claim 1, wherein the nucleic acid molecule is a single stranded nucleic acid template.

3. The method of claim 1, wherein the 5′

end of the nucleic acid molecule is linked to a solid support.

4. The method of claim 1, wherein the solid support comprises a bead, particle, microparticle, microsphere, slide, flowcell or reaction chamber.

5. The method of claim 1, wherein the nucleic acid molecule includes a scissile linkage.

6. The method of claim 1, wherein the nick is introduced into a portion of the nucleic acid molecule with a nicking enzyme or restriction enzyme.

7. The method of claim 1, wherein the nick is introduced by nicking a scissile linkage within the nucleic acid molecule, wherein the scissile linkage comprises a phosphorothioate linkage, phosphoramidate linkage, photolabile internucleosidic linkage, apurinic tetrahydrofuran site, or uracil base.

8. The method of claim 1, further comprising:

a) separating the single-stranded extended primer product from the undegraded portion of the nucleic acid molecule;

b) capturing the separated single-stranded extended primer product onto a solid support; and

c) sequencing the separated single-stranded extended primer product.

9. The method of claim 8, wherein the solid support comprises a bead, micro sphere, nanopore, well, trough, groove, channel, flowcell, microwell, slide or other attachment surface.