Sequencing by structure assembly

• US 10,501,791 B2
• Filed: 10/12/2012
• Issued: 12/10/2019
• Est. Priority Date: 10/14/2011
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Church, George M., Terry, Richard C., Vigneault, Frederic, Vigneault, Francois
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US14/350,975
• Publication Number:
  US 20140349294A1
• Time in Patent Office: 2,615 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

A method of sequencing nucleic acids is provided using sequencing by ligation and/or sequencing by hybridization.

Claims

1. A method for identifying two particular nucleotides at different positions in a target nucleic acid sequence of a target polynucleotide, comprising:

(a) hybridizing a sequencing primer to a single stranded nucleic acid template derived from said target polynucleotide, wherein said single stranded nucleic acid template is immobilized to a support, and wherein said single stranded nucleic acid template comprises said target nucleic acid sequence;

(b) hybridizing an oligonucleotide probe comprising (i) a template hybridizing nucleic acid sequence having a sequence that is complementary with said target nucleic acid sequence, and (ii) a template nonhybridizing nucleic acid structure that does not have a sequence that is complementary with said target nucleic acid sequence, to said single stranded nucleic acid template, thereby forming a hybridized complex comprising said single stranded nucleic acid template, said sequencing primer, and said oligonucleotide probe;

(c) on said hybridized complex, ligating said template hybridizing nucleic acid sequence of said oligonucleotide probe to said sequencing primer, thereby forming an extended hybridized sequence,

wherein said template nonhybridizing nucleic acid structure is attached to said template hybridizing nucleic acid sequence of said oligonucleotide probe, and

wherein said template nonhybridizing nucleic acid structure comprises a plurality of probe hybridization sites comprising a plurality of pre-determined sequences, wherein each of the probe hybridization sites corresponds to one of specific nucleotides at one of specific positions of said oligonucleotide probe and the specific nucleotides are fully complementary to the target nucleic acid sequence;

(d) subsequent to step (c), detecting a hybridization between a plurality of barcode probes and said plurality of pre-determined sequences on said hybridized complex wherein said plurality of barcode probes comprises a first set of barcode probes and a second set of barcode probes, and wherein said detecting a hybridization between a plurality of barcode probes and said plurality of pre-determined sequences comprises (i) hybridizing a first barcode probe from said first set of barcode probes to a first pre-determined sequence of said plurality of pre-determined sequences, (ii) removing said first set of barcode probes unhybridized to the first pre-determined sequence, (iii) identifying the hybridization between said first barcode probe and the first pre-determined sequence by detecting a detectable moiety of said first barcode probe, (iv) removing said first barcode probe hybridized to said first pre-determined sequence from said first pre-determined sequence, (v) hybridizing a second barcode probe from said second set of barcode probes to a second pre-determined sequence of said plurality of pre-determined sequences, and (vi) identifying the hybridization between said second barcode probe and the second pre-determined sequence by detecting a detectable moiety of said second barcode probe; and

(e) determining identities of two of the specific nucleotides in the specific positions of said oligonucleotide probe based on hybridization results from step (d), thereby identifying the two particular nucleotides at the different positions in said target nucleic acid sequence of said target polynucleotide by the two of the specific nucleotides in the specific positions of said oligonucleotide probe.

2. The method of claim 1, wherein said oligonucleotide probe is a nucleic acid sequence having up to about 100 hybridizable nucleotides.

3. The method of claim 1, wherein said template nonhybridizing nucleic acid structure comprises a recognition site for a cleavage agent and said template nonhybridizing nucleic acid structure is removed by said cleavage agent to generate an extendable terminus on said extended hybridized sequence.

4. The method of claim 3, wherein said cleavage agent is a chemical reactant.

5. The method of claim 3, wherein said template nonhybridizing nucleic acid structure comprises photocleavable linkers which are cleavable by ultraviolet (UV) illumination between wavelengths of about 275 nanometers (nm) to about 375 nm.

6. The method of claim 1, wherein said template nonhybridizing nucleic acid structure is a stem and loop structure.

7. The method of claim 1, wherein said template nonhybridizing nucleic acid structure is a linear structure.

8. The method of claim 1, wherein said detectable moiety is optically detectable.

9. The method of claim 1, wherein said detectable moiety is a fluorescent marker or a luminescent marker.

10. The method of claim 1, wherein said template hybridizing nucleic acid sequence includes six nucleotides.

11. The method of claim 1, wherein the number of the probe hybridization sites on said template nonhybridizing nucleic acid structure is equal to the number of the specific nucleotides at the specific positions of said oligonucleotide probe.

12. The method of claim 1, further comprising removing said template nonhybridizing nucleic acid structure to generate an extendable terminus on said extended sequence.

13. The method of claim 12, further comprising, after said removing said template nonhybridizing nucleic acid structure, repeating steps (b) to (e) with an additional oligonucleotide probe having a template hybridizing nucleic acid sequence having a sequence that is complementary with said target nucleic acid sequence and a template nonhybridizing nucleic acid.

14. The method of claim 1, wherein said detectable moiety is selected from the group consisting of a radioactive moiety, enzyme, prosthetic group, fluorescent marker, luminescent marker, bioluminescent marker, metal particle, protein-protein binding pair, and protein-antibody binding pair.

15. The method of claim 1, further comprising removing said second barcode probe hybridized to said second pre-determined sequence from said second pre-determined sequence, hybridizing a third barcode probe from a third set of barcode probes to a third pre-determined sequence of said plurality of pre-determined sequences, identifying the hybridization between said third barcode probe and the third pre-determined sequence by detecting a detectable moiety of said third barcode probe, and determining identity of an additional nucleotide in a position of said oligonucleotide probe different from the two specific nucleotides in the specific positions of said oligonucleotide probe based on the hybridization between said third barcode probe and the third pre-determined sequence, thereby identifying an additional particular nucleotide at a position in said target nucleic acid sequence of said target polynucleotide different from the two particular nucleotides at the different positions in said target nucleic acid sequence of said target polynucleotide by the additional nucleotide in the position of said oligonucleotide probe different from the two specific nucleotides in the specific positions of said oligonucleotide probe.