In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto

A method of in situ detection of a single nucleotide variation of a target nucleic acid in a sample of cells, comprising:

• (A) contacting the sample, wherein the cells of the sample are fixed and permeabilized, with a single nucleotide variation probe and a neighbor probe, wherein the single nucleotide variation probe comprises:
• (i) a single nucleotide variation probe target anchor segment that can specifically hybridize to a region of the target nucleic acid comprising the single nucleotide variation but does not hybridize to a corresponding wild type or non-targeted single nucleotide variation sequence, and (ii) a single nucleotide variation probe pre-amplifier anchor segment;
• and wherein the neighbor probe comprises:
• (i) a neighbor probe target anchor segment that can hybridize to a region of the target nucleic acid adjacent to the binding site of the single nucleotide variation probe, and (ii) a neighbor probe pre-amplifier anchor segment;
• (B) contacting the sample with a single nucleotide variation probe pre-amplifier and a neighbor probe pre-amplifier, wherein the single nucleotide variation probe pre-amplifier comprises a segment complementary to the single nucleotide variation probe pre-amplifier anchor segment and further comprises two or more single nucleotide variation probe collaboration anchors, and wherein the neighbor probe pre-amplifier comprises a segment complementary to the neighbor probe pre-amplifier anchor segment and further comprises two or more neighbor probe collaboration anchors;
• (C) contacting the sample with a collaboration amplifier, wherein the collaboration amplifier comprises a first segment complementary to the single nucleotide variation probe collaboration anchor, a second segment complementary to the neighbor probe collaboration anchor, and a third segment comprising a plurality of label amplifier anchor segments, wherein the collaboration amplifier cannot bind stably to either the single nucleotide variation probe collaboration anchor or the neighbor probe collaboration anchor alone;
• (D) contacting the sample with a label probe system, wherein the label probe system comprises a plurality of label amplifiers and a plurality of label probes, wherein each label amplifier comprises:
• (i) a segment complementary to a label amplifier anchor segment of the collaboration amplifier, and (ii) a plurality of label probe anchor segments;
• wherein each label probe comprises a detectable label and a segment that hybridizes to the label probe anchor segment of label amplifier, wherein the aforesaid hybridizations form a signal generating complex comprising a target nucleic acid with the single nucleotide variation, a single nucleotide variation probe, a neighbor probe, a single nucleotide variation probe pre-amplifier, a neighbor probe preamplifier, a plurality of collaboration amplifiers, a plurality of label amplifiers, and a plurality of label probes;
• (E) removing unbound probes, pre-amplifiers and amplifiers from the sample;
• and
• (F) detecting in situ signal from the signal generating complex on the sample, thereby detecting the single nucleotide variation of the target nucleic acid in the sample of cells.