Methods, kits and compositions for the identification of nucleic acids electrostatically bound to matrices
First Claim
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.
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Accused Products
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.
140 Citations
25 Claims
- 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.
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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. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
- Beacon”
Specification