NANOPORE SEQUENCING USING CHARGE BLOCKADE LABELS

• US 20140309144A1
• Filed: 06/27/2014
• Published: 10/16/2014
• Est. Priority Date: 04/10/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wegener, Jeffrey, Turner, Stephen
• Granted Patent: US 8,986,932 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 composition comprising:

a plurality of polymerase enzymes, each complexed with a template nucleic acid, each polymerase enzyme attached to a nanopore or attached proximal to a nanopore, and nucleic acid sequencing reagents including at least one nucleotide analog having 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 blockade label.

2. The composition of claim 1 wherein the blockade label has a net positive charge.

3. The composition of claim 1 wherein the blockade label comprises a polymer of positively charged monomer units.

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

5. The composition of claim 1 wherein the blockade label comprises lysine, arginine, or ornithine.

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

7. The composition of claim 1 wherein the polyphosphate chain comprises 3, 4, 5, 6, 7, 8, or 9 phosphates.

8. The composition of claim 1 wherein the sequencing reagents comprise two or more different nucleotide analogs, each having a different blockade label.

9. The composition of claim 8 wherein different blockade labels each have a different level of net charge.

10. The composition of claim 8 wherein different blockade labels have a difference in length.

11. The composition of claim 8 wherein the blockade labels comprise polymers of charged monomer units, and different blockade labels have different numbers of monomer units.

12. The composition of claim 8 wherein the two or more different nucleotide analogs comprise linkers having different lengths.

13. The composition of claim 1 wherein the nucleic acid sequencing reagents comprises four different nucleotide analogs, each corresponding to the bases A, G, C, and T, or A, C, G, and U.

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

15. A composition comprising:

a plurality of DNA polymerase enzymes, each complexed with a primed template DNA, each DNA polymerase enzyme attached to a protein nanopore, and nucleic acid sequencing reagents including four different nucleotide analogs corresponding to A, G, C, T, or A, G, C, U, each nucleotide analog having 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 blockade label.

16. The composition of claim 15 wherein the different nucleotide analogs have different levels of charge.

17. The composition of claim 15 wherein the different nucleotide analogs have different sizes.

18. The composition of claim 15 wherein different nucleotide analogs have different blockade labels, each having a different a net positive charge.

19. The composition of claim 15 wherein different nucleotide analogs comprise linkers having different lengths.

20. The composition of claim 15 wherein the polymerase enzymes and protein nanopores to which they are attached comprise fusion proteins.