Patent ID: 7033758

Claim:
A method for in situ detection of a nucleic acid analyte within a sample of biological material using bDNA hybridization comprising the steps of: (a) preparing the sample of biological material by: (i) immobilizing the biological material on a substrate, (ii) permeabilizing the substrate-bound biological material by contacting the substrate-bound biological material to a solution containing Proteinase K at a concentration of about 0.5 μg/ml to about 50 μg/ml, (iii) digesting any RNA in the sample with RNase; and (iv) heating the permeabilized biological material to a temperature and for a time period effective to denature any double-stranded DNA; (b) contacting the biological material with a target oligonucleotide probe under hybridizing conditions, wherein at least a portion of the target probe is complementary to at least a portion of the nucleic acid analyte, so that an analyte-target probe complex is formed when the nucleic acid analyte is present in the sample; (c) washing the biological material with a washing fluid comprising a detergent, at a temperature in the range of approximately 21 to 60° C.; and (d) detecting any analyte-target probe complex on the substrate by: (i) contacting the washed substrate and analyte-target probe complex with a preamplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the preamplifier probe is complementary to a portion of the target probe other than the portion of the target probe that is complementary to the nucleic acid analyte, thereby forming an analyte-target probe-preamplifier probe complex when the nucleic acid analyte is present in the sample, (ii) contacting the product of step (d)(i) with an amplifier oligonucleotide probe under hybridizing conditions, wherein a first portion of the amplifier probe is complementary to a second portion of the preamplifier probe, thereby forming an analyte-target probe-preamplifier probe-amplifier probe complex when the nucleic acid analyte is present in the sample, (iii) contacting the product of step (d)(ii) with a label probe comprised of an alkaline phosphatase conjugated (AP-conjugated) oligonucleotide probe under hybridizing conditions, wherein a portion of the label probe binds to a second portion of the amplifier probe, thereby forming an analyte-target probe-preamplifier probe-amplifier probe-label probe complex when the nucleic acid analyte is present in the sample, (iv) labeling the analyte-target probe-preamplifier probe-amplifier probe-label probe complex with a detectable label comprised of Fast Red, and (v) detecting the presence of the label on the substrate, wherein the nucleic acid analyte is selected from the group consisting of DNA, endogenous genes, and segments thereof.