Nanopore-facilitated single molecule detection of nucleic acids

摘要

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.

主权项

1. A method of detecting a first target oligonucleotide in a sample, the method comprising:
combining the sample with a probe molecule, wherein the probe molecule comprises a terminal extension and a sequence that is fully complementary or partially complementary to the first target oligonucleotide, such that the probe molecule and the target oligonucleotide form a probe/target complex; applying a voltage across a nanopore system while the probe/target complex is on a first side of a partition of the nanopore system, the partition including an opening; and analyzing an electrical current pattern in the nanopore system over time, wherein a presence of the first target oligonucleotide in the sample is indicated by a signature electrical current pattern comprising three sequential levels of electrical current, each level having a duration and a current amplitude different from the other levels of the three sequential levels.