Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores

Patent metadata

Field	Value
Publication number	US-12168799-B2
Application number	US-202117496956-A
Country	US
Kind code	B2
Filing date	Oct 8, 2021
Priority date	Jul 25, 2011
Publication date	Dec 17, 2024
Grant date	Dec 17, 2024

Abstract

The invention relates to a new method of sequencing a double stranded target polynucleotide. The two strands of the double stranded target polynucleotide are linked by a bridging moiety. The two strands of the target polynucleotide are separated using a polynucleotide binding protein and the target polynucleotide is sequenced using a transmembrane pore.

First claim

The invention claimed is:

1. A method comprising:
(i) preparing a nucleic acid construct by ligating a polynucleotide bridging moiety to a 3′ terminus of a first strand and a 5′ terminus of a second strand of a double stranded nucleic acid, wherein the first strand of the double stranded nucleic acid comprises a first nucleic acid sequence and the second strand of the double stranded nucleic acid comprises a second nucleic acid sequence that is complementary to the first nucleic acid sequence;
(ii) using a single molecule sequencing apparatus to perform a sequencing reaction that reads through the first strand, the bridging moiety, and the second strand to produce orthogonal proof-reading sequence information for a methylated nucleobase in the first strand of the construct, wherein the movement of the nucleic acid construct through the single molecule sequencing apparatus is controlled by a Phi29 DNA polymerase;
(iii) comparing the sequence information between the first nucleic acid sequence and the second nucleic acid sequence; and
(iv) determining the presence of a methylated nucleobase in the first sequence.

2. The method of claim 1 , wherein the polynucleotide bridging moiety comprises a marker.

3. The method of claim 2 , wherein the marker is an abasic DNA or a sequence specific nucleic acids.

4. The method of claim 2 , wherein the method further comprising obtaining a signal from the marker in sequencing the bridging moiety that is indicative of the first strand having been sequenced.

5. The method of claim 1 , wherein the Phi29 DNA polymerase is not chemically modified.

6. The method of claim 1 , wherein the Phi29 DNA polymerase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6.

7. The method of claim 1 , wherein the preparing comprises synthesizing a complement of the first sequence to produce the double stranded nucleic acid, wherein the complement contains nucleobases selected from adenine, guanine, thymine, uracil, and cytosine.

8. The method of claim 1 , wherein the first nucleic acid sequence is present in a biological sample, wherein the biological sample is obtained or extracted from an organism or a microorganism.

9. The method of claim 1 , wherein the bridging moiety is a hairpin loop.

10. The method of claim 9 , wherein the hairpin loop is 4-100 nucleotides in length.

11. The method of claim 1 , wherein the hairpin loop is 4-8 nucleotides in length.

12. The method of claim 1 , wherein the first nucleic acid sequence is a DNA sequence.

13. The method of claim 1 , wherein the methylated nucleobase is 5-methylcytosine, 5-hydroxymethylcytosine, or methylcytidine.

14. The method of claim 1 , wherein the double-stranded nucleic acid comprises hemi-genomic DNA.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Brown Clive Gavin
• Clarke James
• Hall Graham
• Harper Gavin
• Heron Andrew John
• White James

Classifications

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• G01N27/44717

  Arrangements for investigating the separated zones, e.g. localising zones · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title