Nanopore-facilitated single molecule detection of nucleic acid
AssigneeThe Curators of the University of Missouri
InventorsLi-Qun Gu, Yong Wang, Kai Tian
Life of this patent
Estimating the term…
Abstract
The present invention provides a new and improved oligonucleotide detection method based on the nanopore technology with a probe containing a complementary sequence to the target oligonucleotide and a terminal extension at the probe's 3′ terminus, 5′ terminus, or both termini. The improved nanopore sensor with the probe enables sensitive, selective, and direct detection, differentiation and quantification of target oligonucleotides such as miRNAs. The inventive detection method may also be employed as a non-invasive and cost-effective diagnostic method for cancer detection based on miRNA levels in the patient's blood sample.
Claims36
1-13. (canceled)
A method of detecting an oligonucleotide in a sample with a nanopore system, comprising the steps of:
- a) mixing a sample suspected of comprising a target oligonucleotide and a probe, said probe comprising a center domain with a complementary sequence to said target oligonucleotide and a terminal extension tagged to at least one of its 3′ and 5′ terminals, to produce a sample mixture wherein said central domain of said probe can hybridize to said target oligonucleotide,
- b) applying a voltage to said sample mixture in a cis compartment of a dual chamber nanopore system sufficient to drive translocation of said hybridized probe and target oligonucleotide through a nanopore of said system by an unzipping process, and
- c) analyzing an electrical current pattern in said nanopore system over time, wherein presence of said oligonucleotide in said sample is indicated by at least one signature electrical current block that is distinct from a background electrical current block that occurs with said sample alone or said probe alone.
(canceled)
A method for detecting two or more distinct target oligonucleotides that differ by at least one nucleotide in a sample with a nanopore system, comprising the steps of:
- a) mixing a sample suspected of comprising said distinct target oligonucleotides and a probe, said probe comprising a center domain with a fully or partially complementary sequence to said target oligonucleotides and a terminal extension tagged to at least one of its 3′ and 5′ terminals, to produce a sample mixture wherein said central domain of said probe can hybridize to said target oligonucleotides,
- b) applying a voltage to said sample mixture in a cis compartment of a dual chamber nanopore system sufficient to drive translocation of hybridized probe and target oligonucleotides through a nanopore of said system by an unzipping process, and
- c) analyzing an electrical current pattern in said nanopore system over time, wherein presence of two or more distinct target oligonucleotides in said sample is indicated by two or more distinct signature electrical current blocks.
(canceled)
33-34. (canceled)
40-44. (canceled)
46-49. (canceled)
A kit comprising:
- (a) a probe comprising a center domain with a fully or partially complementary sequence to a target oligonucleotide and a terminal extension tagged to at least one of its 3′ and 5′ terminals, wherein said central domain of said probe can hybridize to the target oligonucleotide;
- and, (b) a nanopore.
56-60. (canceled)
Dependencies are inferred from the claim text.