Method of determining a target polymer in a sample by using a guide polymer
AssigneesOxford Nanopore Technologies PLC, Monash University
InventorsLakmal Nishantha Jayasinghe, Elizabeth Jayne Wallace, Richard Alexander Gutierrez, Michelle Anne Dunstone, Bradley Alan Spicer
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Abstract
The invention relates generally to a method of detecting and/or analysing target polymers, especially target polynucleotides, using a biological pore. The invention also relates to a novel system for carrying out the method. The method has many uses. In particular, the method may be used for diagnosis, detection of polymorphisms and V(D)J repertoire analysis.
Claims33
A method of determining the presence or absence of a target polymer in a sample comprising:
- a) contacting the sample with a guide polymer that (i) binds to a part of the target polymer and (ii) binds to or is attached to a polymer-guided effector protein, wherein the guide polymer and polymer-guided effector protein form a complex with any target polymer present in the sample;
- b) contacting the sample with a membrane comprising a biological pore with an opening that permits translocation of the complex through the pore;
- c) applying a potential difference across the membrane and taking one or more electrical measurements;
- and
- d) monitoring for the presence or absence of an electrical effect resulting from the translocation of the complex through the pore and thereby determining the presence or absence of the target polymer in the sample.
A method according to any one of the preceding claims, wherein the target polymer is a target polynucleotide.
A method according to any one of the preceding claims, wherein the guide polymer is a guide polynucleotide and the polymer-guided effector protein is a polynucleotide-guided effector protein.
A method according to any one of the preceding claims, wherein the guide polymer, the polymer-guided effector protein, or the target polymer, if present, has an anchor capable of coupling to the membrane.
A method according to any one of the preceding claims, wherein step (a) comprises contacting the sample and the pore with two or more guide polymers each of which (i) bind to a part of the target polymer and (ii) bind to or are attached to two or more polymer-guided effector proteins, wherein the two or more guide polymers and two or more polymer-guided effector proteins form two or more complexes with any target polymer present in the sample and step (d) comprises monitoring for the presence or absence of two or more electrical or current effects resulting from the translocation of the two or more complexes through the pore and thereby determining the presence or absence of the target polymer in the sample.
A method according to any one of the preceding claims, further comprising determining the amount of the target polymer or one or more characteristics of the target polymer if present.
A method according to any one of the preceding claims, wherein the pore is a naturally occurring biological pore or a modified version of a naturally occurring biological pore.
A method according to any one of the preceding claims, wherein the membrane is (i) a triblock copolymer membrane or (ii) a solid-state layer and the biological pore is present in a pore in the solid-state layer.
A system for determining the presence or absence of a target polymer in a sample comprising:
- c) a guide polymer that (i) binds to a part of the target polymer if present and (ii) binds to or is attached to a polymer-guided effector protein, wherein the guide polymer and polymer-guided effector protein form a complex with any target polymer present in the sample;
- and
- d) a biological pore with an opening that permits translocation of the complex through the pore.
Dependencies are inferred from the claim text.