US 10,480,024 B2Granted

Single nucleotide detection method

AssigneesBase4 Innovation Ltd, United Kingdom Research and Innovation

InventorsCameron Alexander Frayling, Barnaby Balmforth, Bruno Flavio Nogueira de Sousa Soares, Thomas Henry Isaac, Boris Breiner, Alessandra Natale, Michele Amasio, Paul Dear

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Abstract

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element.

Claims50

Dependent claims

A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of:

(1) generating a stream of single nucleotide bases from the polynucleotide analyte, wherein the stream of single nucleotide bases is obtained by progressive pyrophosphorolysis of the polynucleotide analyte;
(2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base;
(3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons;
(4) for each captured molecule, labelling the amplicons with a corresponding probe selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base;
(5) for each captured molecule, detecting a property characteristic of the detectable element;
and
(6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte.

4 more elements

A method for determining the sequence of nucleotide bases in a polynucleotide analyte, the method comprising steps of:

(1) generating a stream of single nucleotide bases from the polynucleotide analyte;
(2) producing captured molecules by reacting each single nucleotide base with a capture system, wherein the capture system comprises different sets of capture molecules, each set being selective for a different nucleotide base, and wherein the capture system is comprised of two components for each different nucleotide base:
(a) a first oligonucleotide comprising a double-stranded region and a single-stranded region and (b) a second oligonucleotide that is single-stranded and whose nucleotide base sequence is at least partially complementary to that of the single-stranded region of the first oligonucleotide one nucleotide base beyond the end of the double-stranded region;
(3) for each captured molecule, amplifying at least part of the captured molecule to produce a plurality of amplicons;
(4) for each captured molecule, labelling the amplicons with a corresponding probe that is selective for the amplicons and comprising a different detectable element, wherein the probes are present in a mixture of probes each selective for a different nucleotide base;
(5) for each captured molecule, detecting a property characteristic of the detectable element;
and
(6) from the totality of the results of step (5), determining the sequence of nucleotide bases in the polynucleotide analyte.

5 more elements

Dependencies are inferred from the claim text.