METHODS FOR DETECTING NUCLEIC ACIDS

摘要

Disclosed herein are methods for detecting a target nucleic acid molecule in a sample. The methods can include contacting a sample with a detectably labeled probe (detection probe) that specifically binds to a first target sequence in the target nucleic acid molecule, a bifunctional oligonucleotide including a portion that specifically binds to a second target sequence in the target nucleic acid molecule and a portion that specifically binds to an anchor, and a surface comprising the anchor. Specifically bound detection probe and bifunctional oligonucleotide are ligated and a reagent that specifically removes substantially all of the target nucleic acid is added. Unligated detection probe is removed and presence of the detectable label is detected. In other embodiments, the ligation and/or removal of target nucleic acid are omitted and the detection probe specifically bound to the target nucleic acid is detected.

主权项

1. A method of detecting the presence of a target nucleic acid molecule in a sample, comprising:
a) contacting the sample with:
i) at least one oligonucleotide detection probe comprising a detectable label, wherein the detection probe specifically binds to a first target sequence of the target nucleic acid molecule;ii) at least one bifunctional oligonucleotide that comprises:
A) a target-specific portion, which specifically binds to a second target sequence of the target nucleic acid molecule that is contiguous to the first target sequence; andB) an anchor-specific portion, which specifically binds to an anchor; andiii) at least one surface comprising the anchor, which is immobilized on the surface; under conditions sufficient for:
I) the detection probe to specifically bind to the first target sequence;II) the target-specific portion of the bifunctional oligonucleotide to specifically bind to the second target sequence; andIII) the anchor to specifically bind to the anchor-specific portion of the bifunctional oligonucleotide, such that the target nucleic acid, the detection probe, and the bifunctional oligonucleotide are directly or indirectly bound to the surface via the anchor, thereby producing a tethered target nucleic acid, a tethered detection probe, and a tethered bifunctional oligonucleotide, wherein one or more of steps i), ii), and iii) may occur contemporaneously or sequentially; b) adding a ligase to the tethered detection probe and the tethered bifunctional oligonucleotide, thereby producing a ligated detection probe and bifunctional oligonucleotide; c) adding a reagent to specifically remove substantially all tethered target nucleic acid molecule, wherein the reagent has substantially no specific activity to remove the ligated detection probe and bifunctional oligonucleotide; d) removing substantially all unligated detection probe; and e) detecting on the at least one surface the presence of the detectable label at the position of the anchor, thereby detecting presence of the target nucleic acid molecule in the sample.