Novel nucleic acid template structure for sequencing

Patent metadata

Field	Value
Publication number	US-2024209414-A1
Application number	US-202117905784-A
Country	US
Kind code	A1
Filing date	Mar 10, 2021
Priority date	Mar 11, 2020
Publication date	Jun 27, 2024
Grant date	—

Abstract

Disclosed is a novel structure of a nucleic acid template and the method of making and using the structure. The structure consists of a double-stranded circle with a single-stranded gap. The circular gapped structure includes an extendable end from which copying or sequencing can be initiated.

First claim

1 . A method of forming a gapped circle nucleic acid template, wherein the method comprises the following steps:
(a) attaching an adaptor to at least one end of a double-stranded nucleic acid in a sample forming an adapted nucleic acid, wherein only one strand of the adaptor comprises a cleavage site; (b) joining the ends of the adapted nucleic acid to form a circular adapted nucleic acid; and (c) contacting the circular adapted nucleic acid with a cleaving agent recognizing the cleavage site to remove a portion of only one strand in the circular adapted nucleic acid, thereby forming a gapped circle nucleic acid template having a circular strand and a gapped strand.

2 . The method of claim 1 , wherein the adaptor is attached by extending a primer comprising a target-specific sequence and the adaptor sequence.

3 . The method of claim 1 , wherein the adaptor is attached by ligation.

4 . The method of claim 1 , wherein the adaptor comprises a nucleic acid barcode.

5 . The method of claim 1 , wherein the cleaving agent is a nicking endonuclease and the cleavage site is the nicking endonuclease recognition site.

6 . The method of claim 1 , further comprising a step of amplifying the adapted nucleic acid prior to forming the circular adapted nucleic acid.

7 . The method of claim 1 , wherein the cleaving agent is uracil-N-Glycosylase and the cleavage site is a uridine-containing nucleotide.

8 . The method of claim 1 , further comprising a step of contacting the sample with an exonuclease after the step of forming the circular adapted nucleic acid.

9 . The method of claim 1 , wherein in step (b), joining the ends of the adapted nucleic acid to form a circular adapted nucleic acid is by ligation.

10 . The method of claim 1 , wherein in step (c), removing the portion of only one strand in the circular adapted nucleic acid is by heat denaturation after cleavage with the cleaving agent.

11 . The method of claim 1 , wherein the circular strand comprises a primer-binding site in the gap portion of the gapped circle.

12 . The method of claim 1 , wherein the gapped strand of the gapped circle comprises an extendable 3′-end.

13 . The method of any one of claims 1-12 , further comprising sequencing the target nucleic acid by extending the extendable 3′-end to copy at least a portion of the circular strand.

14 . A method of sequencing nucleic acids in a sample, wherein the method comprises the following steps:
(a) forming a library of gapped circle nucleic acid templates, wherein the method comprises the following steps:
(i) attaching an adaptor to at least one end of double stranded nucleic acids in a sample, thereby forming adapted nucleic acids, wherein only one strand of the adaptor comprises a cleavage site and the adaptor comprises a primer binding site;
(ii) joining the ends of each of the adapted nucleic acids to form circular adapted nucleic acids; and
(iii) contacting the circular adapted nucleic acids with a cleaving agent that recognizes the cleavage site to remove a portion of only one strand in each of the circular adapted nucleic acids, thereby forming a library of gapped circle nucleic acid templates having a gapped strand with an extendable 3′-end and a circular strand; and
(b) extending the extendable 3′-end to copy at least a portion of the circular strand, thereby sequencing the library of gapped circle nucleic acid templates by a sequencing-by-synthesis method.

15 . A method of forming a library of gapped circle nucleic acid templates, wherein the method comprises the following steps:
(a) attaching an adaptor to at least one end of double-stranded nucleic acids in a sample, thereby forming adapted nucleic acids, wherein one strand of the adaptor comprises a cleavage site; (b) joining the ends of each of the adapted nucleic acids to form circular adapted nucleic acids; and (c) contacting the circular adapted nucleic acids with a cleaving agent that recognizes the cleavage site to remove a portion of only one strand in each of the circular adapted nucleic acids, thereby forming a library of gapped circle nucleic acid templates.

16 . A method of forming an enriched library of gapped circle nucleic acid templates, wherein the method comprises the following steps:
(a) attaching an adaptor to at least one end of double stranded nucleic acids in a sample, thereby forming adapted nucleic acids, (b) hybridizing to the adapted nucleic acids a first target-specific primer, wherein the first target-specific primer has a capture moiety; (c) capturing the adapted nucleic acid hybridized to the first primer via the capture moiety, thereby enriching the target nucleic acids; (d) hybridizing to the enriched adapted target nucleic acids a second primer, wherein the second primer comprises a sequence of one or more cleavage sites; (e) extending the second primer to form a double-stranded adapted nucleic acid with one or more cleavage sites on only one strand; (f) joining the ends of each of the double-stranded nucleic acids to form circular adapted nucleic acids; and (g) contacting the circular adapted nucleic acids from step (f) with a cleaving agent that recognizes the cleavage site to remove a portion of only one strand in each of the circular adapted nucleic acids, thereby forming a library of enriched gapped circle nucleic acid templates.

17 . A method of forming an enriched library of gapped circle nucleic acid templates, wherein the method comprises the following steps:
(a) attaching an adaptor to at least one end of double stranded nucleic acids in a sample, thereby forming adapted nucleic acids; (b) hybridizing a first target-specific primer to the adapted nucleic acids, wherein the first target-specific primer comprises a capture moiety; (c) capturing the adapted nucleic acid hybridized to the first primer via the capture moiety; (d) hybridizing a second primer to the captured adapted nucleic acid, wherein the second primer hybridizes to the same strand as the first primer; (e) extending the hybridized second primer, thereby producing a double-stranded adapted nucleic acid and displacing the first primer comprising the capture moiety; (f) hybridizing a third primer to the adaptor within the adapted nucleic acids hybridized to the second primer, wherein the third primer comprising a sequence of one or more cleavage sites; (g) extending the third primer, thereby forming a double-stranded adapted nucleic acid with one or more cleavage sites; (h) joining the ends of each of the double-stranded adapted nucleic acid with one or more cleavage sites to form circular adapted nucleic acids; and (i) contacting the circular adapted nucleic acids from step (h) with a cleaving agent that recognizes the cleavage site to remove a portion of one strand in each of the circular adapted nucleic acids, thereby forming a library of enriched gapped circle nucleic acid templates.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• AYER Aruna
• Chiou Ni-Ting

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title