Methods, Kits and Compositions for the Identification of Nucleic Acids Electrostatically Bound to Matrices

Abstract

This invention pertains to methods, kits and compositions suitable for the detection, identification and/or quantitation of nucleic acids which are electrostatically immobilized to matrices using non-nucleotide probes which sequence specifically hybridize to one or more target sequences of the nucleic acid but do not otherwise substantially interact with the matrix. Once the nucleic acid is immobilized, the detectable non-nucleotide probe/target sequence complex, formed before or after the immobilization of the nucleic acid, can be detected, identified or quantitated under a wide range of assay conditions as a means to detect, identify or quantitate the target sequence in the sample. Because it is reversibly bound, the non-nucleotide probe/target sequence can optionally be removed from the matrix for detecting, identifying or quantitating the target sequence in the sample. Because the non-nucleotide probe/target sequence is protected against degradation, it is another advantage of this invention that the sample can be treated with enzymes which degrade sample components, either before or after the nucleic acid is bound to the matrix, in order to “clean up” the sample (e.g. a complex biological sample such as a cell lysate) and thereby improve the detection, identification or quantitation of the target sequence in the sample. The methods, kits and compositions of this invention are therefore particularly well suited for the analysis, and particularly single point mutation analysis, in a particle assay, in an array assay, in a nuclease digestion/protection assay and/or in a line assay format. When utilized in combination with non-nucleotide “Beacon” probes, the invention is particularly well suited for use in a self-indicating assay format.

1 . A kit for the analysis of a sample containing a nucleic acid molecule comprising a target sequence, said kit comprising a matrix and at least one non-nucleotide probe having a probing nucleobase sequence that sequence specifically hybridizes, under suitable hybridization conditions, to at least a portion of the target sequence sought to be detected in said sample to thereby form a non-nucleotide probe/target sequence complex and wherein the backbone of the non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions, that it exhibits little or no affinity for the matrix. 
     
     
         2 . The kit of  claim 1 , further comprising one or more reagents suitable for modulating the electrostatic binding conditions of the assay. 
     
     
         3 . The kit of  claim 1 , further comprising enzymes that degrade sample contaminants but not the non-nucleotide probe/target sequence complex. 
     
     
         4 . The kit of  claim 1 , wherein the components of the kit are selected to detect organisms in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. 
     
     
         5 . The kit of  claim 1 , wherein the components of the kit are selected to test raw materials, products or processes. 
     
     
         6 . The kit of  claim 1 , wherein the components of the kit are selected to examine clinical samples such as clinical specimens or equipment, fixtures and products used to treat humans or animals. 
     
     
         7 . The kit of  claim 1 , wherein the components of the kit are selected to detect a target sequence that is specific for a genetically-based disease or is specific for a predisposition to a genetically-based disease. 
     
     
         8 . The kit of  claim 1 , wherein the components of the kit are selected detect a target sequence in a forensic technique such as prenatal screening, paternity testing, identity confirmation or crime investigation. 
     
     
         9 . The kit of  claim 1 , wherein the components of the kit are selected to perform a homogeneous assay. 
     
     
         10 . The kit of  claim 1 , wherein the matrix is provided in a form suitable for performing a lateral flow assay. 
     
     
         11 . The kit of  claim 1 , wherein the matrix is provided in a form suitable for performing a line assay. 
     
     
         12 . The kit of  claim 1 , wherein the kit comprises at least two independently detectable non-nucleotide probes and comprises components selected to perform a multiplex assay. 
     
     
         13 . A kit for the analysis of a sample containing a nucleic acid molecule comprising a target sequence, said kit comprising a matrix and at least one non-nucleotide “Beacon” probe having a probing nucleobase sequence that sequence specifically hybridizes, under suitable hybridization conditions, to at least a portion of the target sequence sought to be detected in said sample to thereby form a non-nucleotide “Beacon” probe/target sequence complex and wherein the backbone of the non-nucleotide probe or probes is sufficiently neutral or positively charged, under electrostatic binding conditions, that it exhibits little or no affinity for the matrix. 
     
     
         14 . The kit of  claim 13 , further comprising one or more reagents suitable for modulating the electrostatic binding conditions of the assay. 
     
     
         15 . The kit of  claim 13 , further comprising enzymes that degrade sample contaminants but not a non-nucleotide probe/target sequence complex. 
     
     
         16 . The kit of  claim 13 , wherein the components of the kit are selected to detect organisms in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. 
     
     
         17 . The kit of  claim 13 , wherein the components of the kit are selected to test raw materials, products or processes. 
     
     
         18 . The kit of  claim 13 , wherein the components of the kit are selected to examine clinical samples such as clinical specimens or equipment, fixtures and products used to treat humans or animals. 
     
     
         19 . The kit of  claim 13 , wherein the components of the kit are selected to detect a target sequence that is specific for a genetically-based disease or is specific for a predisposition to a genetically-based disease. 
     
     
         20 . The kit of  claim 13 , wherein the components of the kit are selected to detect a target sequence in a forensic technique such as prenatal screening, paternity testing, identity confirmation or crime investigation. 
     
     
         21 . The kit of  claim 13 , wherein the components of the kit are selected to perform a homogeneous assay. 
     
     
         22 . The kit of  claim 13 , wherein the kit comprises at least two independently detectable non-nucleotide “Beacon” probes and comprises components selected to perform a multiplex assay. 
     
     
         23 . The kit of  claim 22 , wherein the components are selected to perform a multiplex self-indicating assay. 
     
     
         24 . The kit of  claim 13 , wherein the matrix is provided in a form suitable for performing a lateral flow assay. 
     
     
         25 . The kit of  claim 13 , wherein the matrix is provided in a form suitable for performing a line assay.