Nucleic acid sequences using tags

• US 10,526,647 B2
• Filed: 03/03/2017
• Issued: 01/07/2020
• Est. Priority Date: 11/09/2012
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Kalachikov, Sergey, Ju, Jingyue, Morozova, Irina, Dorwart, Michael
• Primary Examiner(s): Woolwine, Samuel C
• Application Number:
  US15/449,757
• Publication Number:
  US 20170241948A1
• Time in Patent Office: 1,040 Days
• Field of Search: None
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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

This disclosure provides chips, systems and methods for sequencing a nucleic acid sample. Tagged nucleotides are provided into a reaction chamber comprising a nanopore in a membrane. An individual tagged nucleotide of the tagged nucleotides can contain a tag coupled to a nucleotide, which tag is detectable with the aid of the nanopore. Next, an individual tagged nucleotide of the tagged nucleotides can be incorporated into a growing strand complementary to a single stranded nucleic acid molecule derived from the nucleic acid sample. With the aid of the nanopore, a tag associated with the individual tagged nucleotide can be detected upon incorporation of the individual tagged nucleotide. The tag can be detected with the aid of the nanopore when the tag is released from the nucleotide.

Claims

1. A method for assembling a protein having a plurality of subunits, the method comprising:

a) providing a plurality of first subunits;

b) providing a plurality of second subunits, wherein the second subunits are modified with respect to the first subunits;

c) contacting the first subunits with the second subunits in a first ratio to form a plurality of proteins having the first subunits and the second subunits, wherein the plurality of proteins have a plurality of ratios of the first subunits to the second subunits; and

d) fractionating the plurality of proteins using ion-exchange chromatography to enrich proteins that have a second ratio of the first subunits to the second subunits, wherein the second ratio is one second subunit per (n−

1) first subunits, wherein ‘

n’

is the number of subunits comprising the protein.

2. The method of claim 1, wherein the protein is a nanopore.

3. The method of claim 2, wherein the nanopore is at least 80% homologous to alpha-hemolysin.

4. The method of claim 1, wherein the first subunits or the second subunits comprise a purification tag.

5. The method of claim 4, wherein the purification tag is a poly-histidine tag.

6. The method of claim 1, wherein the second ratio is 1 second subunit per 6 first subunits.

7. The method of claim 1, wherein the second subunits comprise a chemically reactive moiety.

8. The method of claim 7, wherein the method further comprises (e) performing a reaction to attach an entity to the chemically reactive moiety.

9. The method of claim 8, wherein the protein is a nanopore and the entity is a polymerase.

10. The method of claim 1, wherein the first subunits are wild-type.

11. The method of claim 1, wherein the first subunits and/or second subunits are recombinant.

12. The method of claim 1, wherein the first ratio is approximately equal to the second ratio.

13. The method of claim 1, wherein the first ratio is greater than the second ratio.

14. The method of claim 1, further comprising inserting the proteins having the second ratio of subunits into a bilayer.

15. The method of claim 1, further comprising sequencing a nucleic acid molecule with the aid of the proteins having the second ratio subunits.

16. A nanopore comprising a plurality of subunits, wherein a polymerase is attached to one of the subunits and at least one and less than all of the subunits comprise a first purification tag, and wherein the first purification tag is on the subunits not having the polymerase attached.

17. The nanopore of claim 16, wherein the nanopore is at least 80% homologous to alpha-hemolysin.

18. The nanopore of claim 16, wherein at least one of the subunits comprises a first purification tag, and wherein all of the remaining subunits comprise a first purification tag or a second purification tag.

19. The nanopore of claim 16, wherein the first purification tag is a poly-histidine tag.