Nanopore sequencing methods

• US 10,480,027 B2
• Filed: 01/12/2018
• Issued: 11/19/2019
• Est. Priority Date: 06/08/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Korlach, Jonas
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/869,416
• Publication Number:
  US 20180201993A1
• Time in Patent Office: 676 Days
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 33/48721 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

Abstract

Methods are provided for sequencing of nucleic acid templates using nanopores. A polymerase-nucleic acid complex having a strand displacing polymerase is provided. The nucleic acid in the polymerase-nucleic acid complex is a circular nucleic acid template. Components required for nucleic acid synthesis are provided whereby the polymerase produces a nascent strand that passes through the nanopore while the nascent strand is produced. Current through the nanopore over time is measured allowing for the determination of the sequence of the nascent strand as it translates through the pore using the measured current over time. Because the polymerase can proceed around the circular template multiple times, multiple reads corresponding to the same sequence in the circular template can be obtained in one sequencing process. Where modified bases are present in the template, the method can be used to determine the presence and position of the modified bases using the kinetics of the polymerase.

Claims

1. A method for sequencing a nucleic acid and determining the presence and position of a modified base in a template nucleic acid using a nanopore comprising:

providing a substrate having an upper solution above the substrate and a lower solution below the substrate, the substrate comprising a nanopore connecting the upper solution and lower solution, the nanopore sized to pass a single stranded nucleic acid;

providing a voltage across the nanopore to produce a measurable current flow through the nanopore;

providing, in the upper solution, a polymerase-nucleic acid complex comprising a strand displacing polymerase and a circular nucleic acid template which may have modified bases and providing the components required for nucleic acid synthesis, whereby the polymerase produces a nascent strand that passes through the nanopore as the nascent strand is produced;

measuring the current through the nanopore over time as the nascent strand is translated through the nanopore;

determining a sequence of the nascent strand as it translates through the nanopore using the measured current over time, and determining the presence of a modified base in the circular nucleic acid template using a measured change in kinetics of the polymerase, whereby the polymerase proceeds around the circular template, then continues to produce nascent strand such that a sequence in the nascent strand and the presence of a modified base in the circular template is determined more than once, and

determining the position of the modified base using a determined value for the number of bases between the active site and the bases within the nanopore.

2. The method of claim 1 wherein the polymerase enzyme is attached to the substrate comprising the nanopore.

3. The method of claim 2 wherein the polymerase enzyme is attached proximal to the nanopore.

4. The method of claim 1 wherein the nanopore comprises a biological nanopore.

5. The method of claim 4 wherein the biological nanopore comprises an alpha hemolysin or an MspA protein.

6. The method of claim 4 wherein the polymerase is attached covalently to the biological nanopore.

7. The method of claim 1 wherein the nanopore comprises a solid state nanopore.

8. The method of claim 1 wherein the polymerase proceeds around the circular template more than twice.

9. The method of claim 1 wherein the nucleotide comprises DNA.

10. The method of claim 1 wherein the circular template comprises a double-stranded DNA portion having hairpin loops on each end of the double strand portion.

11. The method of claim 1 wherein the modified bases include methylated bases.

12. The method of claim 1 wherein the modified bases include one or more of 5-methylcytosine, N6-methyladenosine, N3-methyladenosine, N7-methylguanosine, 5-hydroxymethylcytosine, pseudouridine, thiouridine, isoguanosine, isocytosine, dihydrouridine, queuosine, wyosine, inosine, triazole, diaminopurine, β

-D-glucopyranosyloxymethyluracil, 8-oxoguanosine, or 2′

-O-methyl adenosine, 2′

-O-methyl cytidine, 2′

-O-methyl guanosine, or 2′

-O-methyl uridine.

13. The method of claim 1 wherein the modified bases include 5-methylcytosine.

14. The method of claim 1 wherein the circular nucleic acid template comprises natural or genomic DNA.

15. The method of claim 1 wherein either capacitance or electron tunneling current are also measured.

16. The method of claim 1 wherein current oscillation is also measured.

17. The method of claim 1 wherein the polymerase comprises a Phi29 polymerase.

18. The method of claim 1 wherein the value for the number of bases between the active site and the bases within the nanopore is determined by sequencing and simultaneously determining kinetics of the base modification using a known sample.

19. The method of claim 1 wherein the change in kinetics results in a kinetic signature characteristic of the modified base present comprising a pattern of kinetic changes.

20. The method of claim 18 wherein the pattern of kinetic changes comprises 2, 3, 4, 6, 7, 8, or more kinetic changes.