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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. patent application Ser. No. 09 / 456,73 filed on Dec. 8, 1999. This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60 / 111,439 filed on Dec. 8, 1998.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]This invention is related to the field of probe-based detection, analysis and quantitation of nucleic acids which are electrostatically immobilized to matrices. The methods, kits and compositions of this invention are 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, in a line assay and / or in a self-indicating assay format.[0004]2. Description of the Related Art[0005]Nucleic acid hybridization is a fundamental process in molecular biology. Probe-based assays are useful in the detection, quantitation and analysis of nucleic acids. Nucleic...

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Benefits of technology

[0025]In yet another embodiment, this invention relates to a method for the detection, identification or quantitation of a target sequence of a nucleic acid molecule electrostatically immobilized at a location on an array wherein the array comprises nucleic acid molecules electrostatically bound at unique locations. One exemplary method comprises contacting the array with at least one non-nucleotide probe, wherein the non-nucleotide probe will hybridize, under suitable hybridization conditions, to at least a portion of the target sequence if present on the array. The non-nucleotide probe / target sequence complex electrostatically bound at a location on said array is then detected, identified or quantitated as the means to determine the presence, absence or amount of target sequence present at said array location. It is an advantage of the invention that one or more enzymes capable degrading sample contaminates, including the nucleic acid target molecule but not the non-nucleotide probe / target sequence complex, can also be added before analysis of the array to thereby improve the performance of the array assay by degrading sample contaminates which might otherwise lead to false positive results. Optionally, the detectable non-nucleotide probe / target sequence hybrids can be released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe / target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate target sequence in the sample. If the non-nucleotide probes are independently detectable, the analysis of the matrix can proceed in a multiplex format.

[0026]In yet a further embodiment, this invention is directed to a method for the detection, identification or quantitation of a target sequence of a nucleic acid molecule which may be present in any of two or more samples of interest. The method comprises mixing each of the two or more samples of interest with at least one non-nucleotide probe, under suitable hybridization conditions. Next a matrix is contacted, under suitable electrostatic binding conditions, with at least a portion of each of the two or more samples to thereby electrostatically immobilize the nucleic acid components of each sample to the matrix, each at a unique location, and thereby create a matrix array of samples. The non-nucleotide probe / target sequence complex electrostatically bound at a location on said array is then detected, identified or quantitated as the means to determine the presence, absence or amount of target sequence present at said array location. It is an advantage of the invention that one or more enzymes capable degrading sample contaminates, possibly including the nucleic acid target molecule but not the non-nucleotide probe / target sequence complex, can also be added before analysis of the array to thereby improve the performance of the array assay by degrading sample contaminates which might otherwise lead to false positive results. Optionally, the detectable non-nucleotide probe / target sequence hybrids can be released from the matrix by adjustment of conditions outside the range required for electrostatic binding and thereby facilitates detection of the unbound non-nucleotide probe / target sequence hybrid, or just the detectable probe, as the means to detect, identify or quantitate target sequence in the sample. If the non-nucleotide probes are independently detectable, the analysis of the matrix can proceed in a multiplex format.

[0027]In yet another embodiment, this invention is directed to kits suitable for performing an assay which detects the presence, absence or number of target sequences present in a sample. The kits of this invention comprise a matrix and one or more non-nucleotide probes and optionally one or more other reagents or compositions which are selected to perform an assay of this invention or otherwise simplify the performance of an assay used to detect, identify or quantitate a target sequence in a sample.

[0028]The compositions, methods and kits of this invention are particularly useful for the detection, identification and / or enumeration of bacteria and eucarya (e.g. pathogens) in food, beverages, water, pharmaceutical products, personal care products, dairy products or environmental samples. The analysis of preferred non-limiting beverages include soda, bottled water, fruit juice, beer, wine or liquor products. Suitable compositions, methods and kits will be particularly useful for the analysis of raw materials, equipment, products or processes used to manufacture or store food, beverages, water, pharmaceutical products, personal care products dairy products or environmental samples.

[0029]Additionally, the compositions, methods and kits of this invention are particularly useful for the detection of bacteria and eucarya (e.g. pathogens) in clinical samples and clinical environments. Suitable compositions, methods and kits will be particularly useful for the analysis of clinical specimens, equipment, fixtures or products used to treat humans or animals.

Problems solved by technology

This requirement for substantial optimization of assay conditions within a very narrow operating range results because electrostatic immobilization of nucleic acid is a relatively non-specific process and therefore it is difficult to electrostatically immobilize a negatively charged target nucleic acid to a cationic surface without the positively charged matrix also exhibiting a strong affinity for the negatively charged nucleic acid probe.

Consequently, the applicability of the assays of Arnold et al. are of limited practical utility.

ses. However, a substantial limitation of the Gerdes et al. invention is that the nucleic acid must be irreversibly bound to the highly electropositive solid phase mate

Furthermore, Shuber does not suggest, disclose or teach any advantages, such the ability to work within a broad range of assay conditions, of performing a peptide nucleic acid-based analysis of nucleic acid electrostatically immobilized to a matrix.

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