NANOPORE SEQUENCING USING CHARGE BLOCKADE LABELS

• US 20130240359A1
• Filed: 05/14/2013
• Published: 09/19/2013
• Est. Priority Date: 04/09/2010
• 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, Wegener, Jeffrey
• Granted Patent: US 8,652,779 B2
• Time in Patent Office: Days
• US Class Current: 204/451
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• 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

The invention relates to devices and methods for nanopore sequencing. The invention includes compositions and methods of nucleic acid sequencing using a single polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid attached proximal to a nanopore, and nucleotide analogs in solution comprising charge blockade label that are attached to the polyphosphate portion of the nucleotide analog such that the charge blockade labels are cleaved when the nucleotide analog is incorporated into a growing nucleic acid and the charge blockade label is detected by the nanopore to determine the presence and identity of the incorporated nucleotide and thereby determine the sequence of a template nucleic acid.

Claims

1. A method for determining sequence information about at template nucleic acid molecule comprising

providing a substrate having at least one nanopore, the at least one nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid;

contacting the substrate with a sequencing reaction mixture comprising reagents required for polymerase mediated nucleic acid synthesis including two or more different types of nucleotide analogs, each comprising a current blockade label attached to the phosphate portion of the nucleotide analogs such that the current blockade label is cleaved upon incorporation of the nucleotide into a growing strand,

providing a voltage drop across the nanopore such that the current blockage label produces a change in current through the nanopore;

measuring the current through the nanopore over time to detect the incorporation of nucleotides into the growing strand; and

identifying the type of nucleotide incorporated into the growing strand using current blockage characteristics, thus determining sequence information about the template nucleic acid molecule.

2. The method of claim 1 wherein the sequencing reaction mixture comprises four different types of nucleotide analogs, each corresponding to the bases A, G, C, and T, or A, C, G, and U.

3. The method of claim 2 wherein each of the types of nucleotides has a different blockade label.

4. The method of claim 1 wherein the current blockage characteristics comprise the magnitude of the current through the nanopore.

5. The method of claim 1 wherein the current blockage characteristics comprise the shape of the measured current through the nanopore over time.

6. The method of claim 1 wherein the nucleotide analogs have the structure NS-PP-L-B wherein NS comprise a nucleoside moiety, PP comprises a polyphosphate chain with at least two phosphates, L comprises a linker, and B comprises a charge blockade label.

7. The method of claim 1 wherein the different types of nucleic acids have different current blockade labels, each having a different level of net charge.

8. The method of claim 1 wherein the different types of nucleotide analogs comprise linkers having different lengths.

9. The composition of claim 1 wherein a blockade label comprises a protein.

10. The composition of claim 1 wherein a blockade label comprises lysine, arginine, or ornithine.

11. The composition of claim 1 wherein the linker group comprises polyethylene glycol or a branched or linear alkane.

12. The composition of claim 1 wherein the phosphate portion of a nucleotide analog comprises 3, 4, 5, 6, 7, or 8 phosphates.

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

14. The method of claim 1 wherein the nanopore comprises a nanopore protein.

15. The method of claim 1 wherein the nanopore comprises a hybrid nanopore comprising a solid state pore dimensioned and treated so as to accept a single nanopore protein.

16. The method of claim 1 wherein the polymerase enzyme is attached directly to the substrate.

17. The method of claim 1 wherein the polymerase enzyme comprises a fusion protein between a polymerase enzyme and a nanopore protein.

18. The method of claim 1 wherein the voltage drop across the nanopore is positive on the top side of the nanopore relative to the bottom side of the nanopore, such that positively charged molecules tend to be transported through the nanopore.

19. The method of claim 18 wherein the cleaved portions of the nucleotide analogs have a net negative charge.

20. The method of claim 1 wherein the nanopore has a non-cylindrical shape.

21. The method of claim 1 wherein the nanopore has a truncated cone shape.

22. The method of claim 1 wherein the current is measured using measurements of voltage, resistance, or impedance.

23. The method of claim 1 wherein each of the nucleotide analogs comprise a linker between the base portion and blockade label, whereby when the base portion of the nucleotide analog is associated with the polymerase enzyme while it is incorporated into a growing strand during nucleic acid synthesis, the current blockage label at least partially enters the nanopore, resulting in a measureable change in current through the nanopore while the nucleotide analog is associated with the polymerase, and using the change in current while the nucleotide analog is associated with the polymerase to detect and identify the incorporated nucleotide.

24. The method of claim 23 wherein the different types of nucleotide analogs have linkers, each having a different elastic modulus.

25. The method of claim 1 wherein the top opening and bottom opening of the nanopore are each accessible to the top of the substrate.

26. A system for nucleic acid sequencing comprising:

a substrate having at least one nanopore, the at least one nanopore having a top opening and a bottom opening, and having a single polymerase enzyme attached proximal to an opening, the polymerase enzyme complexed with a primed template nucleic acid;

a sequencing reaction mixture in contact with the substrate comprising reagents required for polymerase mediated nucleic acid synthesis including two or more types of nucleotide analogs, each comprising a current blockade label;

drive electrodes in contact with the reaction mixture on either side of the substrate for producing a voltage drop across the at least one nanopore;

one or more measurement electrodes connected to electronic measurement equipment for measuring the current through the nanopore over time; and

a computer for identifying the type of nucleotide incorporated into the growing strand.

27. The method of claim 26 wherein the sequencing reaction mixture comprises four different types of nucleotide analogs, each corresponding to the bases A, G, C, and T, or A, C, G, and U.