Nuclease protection methods for detection of nucleotide variants

US9765385B2 · kind B2 · utility

Assignee

• HTG Molecular Diagnostics, Inc. · US

Inventors

• Matt Rounseville · Tucson, US
• Bruce Seligmann · Tucson, US

Key dates

• Filing date: Jun 28, 2013
• Grant date: Sep 19, 2017
• Expiry date: Oct 7, 2033

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q2561/108
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

Disclosed herein are methods for detecting presence of a nucleotide variant in a target nucleic acid utilizing a nuclease protection assay. The methods include contacting a sample with at least two probes, wherein the first probe is complementary to the wild-type (non-variant) nucleotide(s) at the nucleotide variant position(s) in the target nucleic acid and the second probe is complementary to the variant nucleotide(s) at the nucleotide variant position(s) in the target nucleic acid, under conditions sufficient for the probes to hybridize to the target nucleic acid, producing a mixture of hybridized and unhybridized nucleic acids. The mixture is contacted with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove unhybridized nucleic acid molecules (or unhybridized portions of nucleic acid molecules). The presence of the at least two probes is then detected, thereby detecting the presence of the variant and/or non-variant target nucleic acid in the sample.