Compositions and methods for polynucleotide sequencing

Patent metadata

Field	Value
Publication number	US-10364462-B2
Application number	US-201715606354-A
Country	US
Kind code	B2
Filing date	May 26, 2017
Priority date	Nov 26, 2013
Publication date	Jul 30, 2019
Grant date	Jul 30, 2019

Abstract

Methods and compositions for characterizing a target polynucleotide, including, characterizing the sequence of the target polynucleotide, using the fractional translocation steps of the target polynucleotide's translocation through a pore.

First claim

What is claimed is:

1. A system for characterizing a target polynucleotide, comprising:
a polypeptide pore inserted into a membrane having a cis and a trans surface;
a Hel308 helicase contacting the pore on the cis surface of the membrane;
a solution comprising a target polynucleotide and less than 1 mM ATP or analogue thereof contacting the cis surface of the membrane;
an amplifier adapted to provide a potential difference across the membrane;
a processor with instructions to characterize signals from fractional step translocations of the polynucleotide through the polypeptide pore; and
a detector adapted to measure an ionic current flowing through the pore.

2. The system of claim 1 , wherein the instructions are adapted to characterize at least two signals during a full translocation cycle of the Hel308 helicase.

3. The system of claim 1 , wherein the instructions comprise a Hidden Markov model.

4. The system of claim 1 , wherein the detector is adapted to measure at least two signals during a full translocation cycle of the Hel308 helicase.

5. The system of claim 1 , wherein the detector measures a current response at a sampling rate of about 50 kHz.

6. The system of claim 1 , wherein the polypeptide pore comprises a constriction zone with a length that can hold no more than five nucleotides.

7. The system of claim 1 , wherein the polypeptide pore comprises a constriction zone with a length that can hold no more than two nucleotides.

8. The system of claim 1 , wherein the solution comprises less than about 100 μM ATP or analogue thereof.

9. The system of claim 1 , wherein the polypeptide pore comprises a Mycobacterium smegmatis porin A (MspA) pore.

10. The system of claim 1 , wherein the polypeptide pore comprises a wild type MspA polypeptide comprising one or more mutations selected from the group consisting of D90N, D91N, D93N, D118R, D134R and E139K.

11. The system of claim 1 , wherein the polypeptide pore comprises an amino acid sequence having at least about 90% homology to SEQ ID NO: 1.

12. The system of claim 1 , wherein the polypeptide pore comprises an amino acid sequence of SEQ ID NO: 1.

13. The system of claim 1 , wherein the Hel308 helicase comprises a motif having an amino acid sequences selected from the group consisting of SEQ ID NO:04-65.

14. The system of claim 1 , wherein the membrane comprises a lipid bilayer.

15. The system of claim 14 , wherein the lipid bilayer comprises 1,2-diphytanoyl-sn-glycero-3-phosphocholine.

16. A method of characterizing a target polynucleotide, the method comprising:
(a) obtaining the system of claim 1 ;
(b) applying a potential difference across a pore in contact with a Hel308 helicase and the target polynucleotide;
(c) measuring at least two signals for a unit of one or more nucleotides of the target polynucleotide moving through the pore during a full translocation cycle of the Hel308 helicase; and
(d) characterizing the target polynucleotide using the at least two signals measured in (c).

17. The method of claim 16 , wherein the at least two signals comprise electrical signals.

18. The method of claim 16 , further comprising repeating steps (b) (d).

19. The method of claim 16 , wherein a full translocation cycle of the Hel308 helicase comprises nucleotide substrate binding and nucleotide substrate hydrolysis.

Assignees

• Illumina Inc

Inventors

• Stava Eric
• Gundlach Jens H
• Mandell Jeffrey G
• Gunderson Kevin L
• Derrington Ian M
• Mohimani Hosein

Classifications

• C07K14/35

  from Mycobacteriaceae (F) · CPC title

• G01N27/447

  using electrophoresis · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2521/513

  Winding/unwinding enzyme, e.g. helicase · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title