Modified base detection with nanopore sequencing

• US 9,116,118 B2
• Filed: 06/10/2013
• Issued: 08/25/2015
• 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): MARSCHEL, ARDIN H
• Application Number:
  US13/914,361
• Publication Number:
  US 20130327644A1
• Time in Patent Office: 806 Days
• Field of Search: 435/6.1
• US Class Current: 1/1
• 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, compositions, and systems are provided for characterization of modified nucleic acids. Nanopore sequencing is performed using a processive enzyme to control the rate of translation of a single strand of a nucleic acid through a nanopore. The presence and identity of a modified base can be determined by monitoring the kinetics of translation through the pore even though the events that lead to the kinetic changes are separated in time and space from the translation of the modified base or it'"'"'s compliment through the nanopore. The invention also comprises the use of hemi-genomic DNA in nanopore sequencing.

Claims

1. A method for sequencing a nucleic acid template and identifying modified bases therein 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;

controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid;

measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation;

determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and

determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme from the interaction of the modified base with the processive enzyme.

2. The method of claim 1 wherein the template nucleic acid is sequenced multiple times.

3. The method of claim 1 wherein the processive enzyme comprises polymerase, exonuclease, or helicase activity.

4. The method of claim 1 wherein the processive enzyme comprises a DNA polymerase.

5. The method of claim 4 wherein the DNA polymerase has 3′

to 5′

exonuclease activity.

6. The method claim 1 wherein the processive enzyme comprises a helicase.

16. The method of claim 1 wherein the substrate comprises from 1,000 to one million nanopores.

17. The method of claim 1 wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand.

18. The method of claim 17 wherein the nascent strand is translated through the nanopore.

19. The method of claim 17 wherein the genomic strand is translated through the nanopore.

20. The method of claim 17 wherein the genomic strand and nascent strand are attached through a hairpin loop and both strands are translated through the nanopore.

7. A method for sequencing a nucleic acid 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;

controlling the rate of translation of a single stranded portion of the template nucleic acid through the nanopore with a processive enzyme associated with a template nucleic acid;

measuring the current through the nanopore over time as it is translated through the nanopore, wherein such measuring includes measuring the current and measuring the rate of translation;

determining the sequence of a portion of the template nucleic acid as it translates through the nanopore using the measured current over time; and

determining the presence of modified bases in the template nucleic acid by correlating changes in the rate of translation of the nucleic acid through the nanopore to changes in the kinetics of the processive enzyme due to the interaction of the modified base with the processive enzyme;

wherein the template nucleic acid comprises hemi-genomic DNA comprising a genomic strand and a nascent strand.

8. The method of claim 7 wherein the nascent strand is translated through the nanopore.

9. The method of claim 7 wherein the genomic strand is translated through the nanopore.

10. The method of claim 7 wherein the genomic strand and nascent strand are attached through a hairpin loop and both strands are translated through the nanopore.

11. The method of claim 7 wherein the processive enzyme comprises polymerase, exonuclease, or helicase activity.

12. The method of claim 7 wherein the processive enzyme comprises a DNA polymerase.

13. The method of claim 12 wherein the DNA polymerase has 3′

to 5′

exonuclease activity.

14. The method of claim 7 wherein the processive enzyme comprises a helicase.

15. The method of claim 7 wherein the template nucleic acid is sequenced multiple times.