Method and products for characterizing a polynucleotide using a nanopore
AssigneeOxford Nanopore Technologies PLC
InventorsLukasz Ciszewski, Sebastien James, Graham James Hall, Olle Alfred Nordesjö
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Abstract
Provided herein are methods of characterising a polynucleotide during the translocation of the polynucleotide through a nanopore. Also provided herein are methods of operating a nanopore array. Further provided herein are kits and apparatuses for carrying out such methods.
Claims35
A method of characterising a polynucleotide, comprising:
- (a) contacting the polynucleotide with a nanopore having a first opening and a second opening;
- (b) translocating a portion of the polynucleotide through the nanopore in a first direction with respect to the nanopore, wherein the first direction is from the first opening to the second opening;
- (c) contacting the translocated portion of the polynucleotide with one or more conditions which decrease formation of cation-mediated secondary structure;
- (d) translocating the polynucleotide in a second direction with respect to the nanopore, wherein the second direction is from the second opening to the first opening;
- and
- (e) taking one or more measurements characteristic of the polynucleotide during the translocation of the polynucleotide through the nanopore, thereby characterising the polynucleotide.
A method according to any one of the preceding claims, wherein the method comprises characterising a plurality of polynucleotides using a plurality of nanopores, optionally wherein said plurality of nanopores are present in a nanopore array.
A method of operating a nanopore array, wherein each nanopore in the array has a first opening and a second opening, the method comprising:
- (a) contacting the array with a plurality of polynucleotides under conditions such that a portion of each polynucleotide translocates through the array in a first direction with respect to the nanopores in the array, wherein the first direction is from the first opening to the second opening;
- (b) contacting the translocated portion of the polynucleotides with one or more conditions which decrease formation of cation-mediated secondary structure;
- (c) determining whether each polynucleotide translocated in step (b) has a desired characteristic;
- and
- (d) ejecting polynucleotides which do not have the desired characteristic by translocating the polynucleotide in a second direction with respect to the nanopores in the array, wherein the second direction is from the second opening to the first opening.
A method according to any one of the preceding claims, wherein the method is performed at a temperature lower than the melting temperature of the cation-mediated secondary structure.
A method according to any one of the preceding claims, wherein the cation-mediated secondary structure is a guanine tetrad or a G-quadruplex, preferably a G-quadruplex.
A method according to any one of the preceding claims, wherein the one or more conditions comprise a metal salt that disrupts the stability of the cation-mediated secondary structure.
A method according to any one of the preceding claims, wherein the conditions are substantially free of K+.
A method according to any one of the preceding claims, wherein the polynucleotide, or each polynucleotide in the plurality of the polynucleotides, has a length of 100 nucleotides or more.
A method according to any one of the preceding claims, wherein the polynucleotide has a length of 5000 nucleotides or more, or the polynucleotides in the plurality of polynucleotides have an average length of 5000 nucleotides or more.
A method according to any one of the preceding claims, comprising controlling the movement of the or each polynucleotide with respect to the nanopore in the first direction using a polynucleotide-handling protein.
A method according to any one of the preceding claims, wherein the nanopore is a protein pore, a solid state pore or a DNA origami pore, preferably wherein the nanopore is a transmembrane protein pore.
A method according to any one of the preceding claims, wherein the one or more characteristics comprises (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.
A method according to any one of the preceding claims, comprising applying a voltage potential across the nanopore via one or more platinum electrodes.
A kit for characterising a polynucleotide, comprising:
- (a) a nanopore in a membrane;
- and
- (b) a solution comprising one or more metal salts selected from the group consisting of one or more rubidium salts, sodium ferricyanide and sodium ferrocyanide.
An apparatus for characterising a polynucleotide, comprising:
- (a) an array of nanopores in a membrane;
- and
- (b) a solution comprising one or more metal salts in contact with the membrane, wherein the one or more metal salts are selected from the group consisting of one or more rubidium salts, sodium ferricyanide and sodium ferrocyanide.
Use of a one or more metal salts for reducing cation-mediated secondary structure in a polynucleotide in a nanopore apparatus or during a method of characterising a polynucleotide using a nanopore, wherein the one or more metal salts are selected from the group consisting of one or more rubidium salts, sodium ferricyanide and sodium ferrocyanide.
Dependencies are inferred from the claim text.