Sequencing polynucleotides using nanopores
Sequencing polynucleotides using nanopores is provided herein. A polynucleotide is disposed through a nanopore's aperture such that its 3′ end is on the nanopore's first side and its 5′ end is on the nanopore's second side. On the nanopore's first side, a duplex with the polynucleotide is formed that includes a 3′ end. The duplex is extended on the first side of the nanopore by adding a nucleotide to the 3′ end of the duplex. A first force is applied disposing the 3′ end of the duplex within the aperture, and the nanopore inhibits translocation of the 3′ end of the duplex to the second side of the nanopore. A value of an electrical property of the 3′ end of the duplex and a single-stranded portion of the polynucleotide is measured. The nucleotide at the 3′ end of the duplex is identified using the measured value.
1 . A method of sequencing a polynucleotide using a nanopore comprising a first side, a second side, and an aperture extending through the first and second sides, the method comprising:
(a) disposing a polynucleotide through the aperture of the nanopore such that a 3′ end of the polynucleotide is on the first side of the nanopore, and a 5′ end of the polynucleotide is on the second side of the nanopore;
(b) hybridizing a primer to a primer binding sequence in the polynucleotide to form a duplex on the first side of the nanopore, the duplex including a 3′ end corresponding to a 3′ end of the primer;
(c) extending the duplex on the first side of the nanopore by adding a natural nucleotide to the 3′ end of the duplex;
(d) applying a force disposing the 3′ end of the extended duplex within the aperture, and while applying the force:
inhibiting, using the nanopore, translocation of the 3′ end of the extended duplex to the second side of the nanopore; and
measuring a value of an electrical property of the 3′ end of the extended duplex and a single-stranded portion of the polynucleotide; and
(e) identifying the natural nucleotide using the value measured in operation (d).
2 . The method of claim 1 , wherein the value measured in operation (d) comprises an electrical current, ionic current, electrical resistance, or electrical voltage drop across the nanopore.
3 . The method of claim 1 , wherein the value measured in operation (d) comprises noise of an electrical current, ionic current, electrical resistance, or electrical voltage drop across the nanopore.
4 . The method of claim 3 , wherein the value measured in operation (d) comprises a standard deviation of the noise.
5 . The method of claim 1 , wherein the value measured in operation (d) is at least based on M nucleotides of the single-stranded portion of the polynucleotide and D pairs of hybridized nucleotides of the extended duplex, wherein M is greater than or equal to two, and wherein D is greater than or equal to one.
6 . The method of claim 5 , wherein M is greater than or equal to three.
7 . The method of claim 5 , wherein D is greater than or equal to two.
8 . The method of claim 5 , wherein at least one of the M nucleotides of the single-stranded portion comprises a modified base, the method comprising identifying the modified base using the value measured in operation (d).
9 . The method of claim 8 , wherein the modified base comprises a methylated base.
10 . The method of claim 1 , further comprising:
(f) applying a modified force again disposing the 3′ end of the extended duplex within the aperture, and while applying the modified force:
inhibiting, using the nanopore, translocation of the 3′ end of the extended duplex to the second side of the nanopore; and
measuring a value of an electrical property of the 3′ end of the extended duplex and a single-stranded portion of the polynucleotide; and
(g) identifying the natural nucleotide using the value measured in operation (f).
11 . The method of claim 1 , further comprising, after operation (d):
dissociating the extended duplex from the polynucleotide; and
hybridizing a second primer to the primer binding sequence of the polynucleotide to form a new duplex with the polynucleotide on the first side of the nanopore, the new duplex including a new 3′ end corresponding to a 3′ end of the second primer polynucleotide.
12 . The method of claim 1 , wherein the nanopore comprises a solid-state nanopore.
13 . The method of claim 1 , wherein the nanopore comprises a biological nanopore.
14 . The method of claim 13 , wherein the biological nanopore comprises MspA.
15 . The method of claim 1 , wherein the polynucleotide comprises RNA.
16 . The method of claim 1 , wherein the polynucleotide comprises DNA.
17 . A sequencing system, comprising:
a nanopore comprising a first side, a second side, and an aperture extending through the first and second sides;
a polynucleotide disposed through the aperture of the nanopore such that a 3′ end of the polynucleotide is on the first side of the nanopore, and a 5′ end of the polynucleotide is on the second side of the nanopore;
a primer hybridized to a primer binding sequence in the polynucleotide to form a duplex disposed on the first side of the nanopore, the duplex including a 3′ end corresponding to a 3′ end of the primer and at which a natural nucleotide is disposed; and
circuitry configured to:
apply a force disposing the 3′ end of the duplex within the aperture;
measure a value of an electrical property of the 3′ end of the duplex and a single-stranded portion of the polynucleotide while applying the force; and
identify the natural nucleotide using the measured value,
wherein the nanopore inhibits translocation of the 3′ end of the duplex to the second side of the nanopore while the force is applied.
18 . The system of claim 17 , wherein the value measured by the circuitry comprises an electrical current, ionic current, electrical resistance, or electrical voltage drop across the nanopore.
19 . The system of claim 17 , wherein the value measured by the circuitry comprises noise of an electrical current, ionic current, electrical resistance, or electrical voltage drop across the nanopore.
20 . The system of claim 19 , wherein the value measured by the circuitry comprises a standard deviation of the noise.
21 . The system of claim 17 , wherein the value measured by the circuitry is at least based on M nucleotides of the single-stranded portion of the polynucleotide and D pairs of hybridized nucleotides of the duplex, wherein M is greater than or equal to two, and wherein D is greater than or equal to one.
22 . The system of claim 21 , wherein M is greater than or equal to three.
23 . The system of claim 21 , wherein D is greater than or equal to two.
24 . The system of claim 21 , wherein at least one of the M nucleotides of the single-stranded portion comprises a modified base, the circuitry being configured to identify the modified base using the value measured by the circuitry.
25 . The system of claim 24 , wherein the modified base comprises a methylated base.
26 . The system of claim 17 , wherein the circuitry further is configured to:
apply a modified force again disposing the 3′ end of the duplex within the aperture;
measure a value of an electrical property of the 3′ end of the duplex and a single-stranded portion of the polynucleotide while applying the modified force; and
identify the natural nucleotide using the measured value,
wherein the nanopore inhibits translocation of the 3′ end of the duplex to the second side of the nanopore while the modified force is applied.
27 . The system of claim 17 , wherein the circuitry is configured to, after applying the force, dissociate the duplex from the polynucleotide.
28 . The system of claim 17 , wherein the nanopore comprises a solid-state nanopore.
29 . The system of claim 17 , wherein the nanopore comprises a biological nanopore.
30 . The system of claim 29 , wherein the biological nanopore comprises MspA.
31 . The system of claim 17 , wherein the polynucleotide comprises RNA.
32 . The system of claim 17 , wherein the polynucleotide comprises DNA.