Highly sensitive gene detection and localization using in situ branched-DNA hybridization
AssigneeBayer Aktiengesellschaft
InventorsDaryn Kenny, Lu Ping Shen, Vincent P. Antao, Audrey N. Player, Wei Cao
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Abstract
Methods are provided for highly sensitive and rapid in situ detection of a nucleic acid analyte of a known sequence. The method employs oligonucleotide probes in a series of optimized steps to amplify a signal and decrease background. Sensitivity is enhanced such that the method can detect as few as 1-2 copies of nucleic acid analyte per sample, the sample containing a cell, tissue or similar biological material. Methods of detecting and identifying the position of the nucleic acid analyte in a cell are also provided.
Claims35
A method for in situ detection of a nucleic acid analyte within a sample of biological material based on 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;
- and
- (iii) optionally 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.
The method of claim 10, wherein the buffer solution comprises the salts of alkali metals.
A method for identifying the position of a nucleic acid analyte within a sample of biological material based on 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;
- and
- (iii) optionally 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.;
- (d) detecting any analyte-target probe complex on the substrate;
- and
- (e) identifying the position of the analyte-target probe complex within a cell of the biological sample as indicative of the position of the nucleic acid analyte in the cell.
A method for detecting a nucleic acid analyte within a sample of biological material comprising performing bDNA hybridization to detect the nucleic acid analyte in situ, wherein the method has a sensitivity sufficient to detect from about 1 to about 10 copies of the nucleic acid analyte in the biological material.
Dependencies are inferred from the claim text.