NUCLEASE PROTECTION METHODS FOR DETECTION OF NUCLEOTIDE VARIANTS

摘要

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.

主权项

1. A method of detecting presence of a nucleotide variant in a target nucleic acid in a sample, comprising:
contacting the sample with at least two probes complementary to the target nucleic acid molecule comprising a nucleotide variant under conditions sufficient for the first probe and the second probe to hybridize to the target nucleic acid, producing a mixture of hybridized nucleic acid molecules and unhybridized nucleic acid molecules, wherein the first probe is complementary to wild type at the target nucleic acid molecule nucleotide variant, wherein the target nucleic acid molecule nucleotide variant position is at least two bases from an end of the first probe, and wherein the second probe is complementary to a first variant for the target nucleic acid molecule nucleotide variant, wherein the target nucleic acid nucleotide variant position is at least two bases from an of the second probe; contacting the mixture of hybridized nucleic acid molecules and unhybridized nucleic acid molecules with a nuclease specific for single-stranded nucleic acid molecules under conditions sufficient to remove the unhybridized nucleic acid molecules; and detecting presence of one or more of the probes in the mixture, thereby detecting presence of the nucleotide variant in the sample.