Methods of sandwich hybridization for the quantitative analysis of oligonucleotides
First Claim
1. A method of quantitative analysis of an oligonucleotide analyte, said analysis using an assay plate prepared with masked receptor sites and two sensitized polynucleotide DNA probes, each having a different sequence complementary to the analyte comprising performing the hereinbelow steps in the order presented:
- (a) attaching the first DNA probe to the receptor sites;
(b) then performing the following two substeps in any order, that is sequentially or in reverse order;
(1) hybridizing the oligonucleotide analyte to the complementary portions of the first and second DNA probes; and
,(2) labeling by the attachment of an indicator to the second DNA probe; and
,(c) detecting the intensity of the indicator and thereby obtaining a quantitation of said oligonucleotide analyte.
1 Assignment
0 Petitions
Accused Products
Abstract
The invention is directed to a sandwich hybridization assay wherein a nucleic acid capture probe is firstly immobilized on an assay plate via masked receptors on the plate. The capture probe is immobilized by the binding of receptor ligands on the capture probe during this first step. Subsequently, target nucleic acid is hybridized to the immobilized capture probe either before or after the hybridization of an indicator nucleic acid probe onto the target. The target is quantified via detection of the immobilized indicator signal.
110 Citations
16 Claims
-
1. A method of quantitative analysis of an oligonucleotide analyte, said analysis using an assay plate prepared with masked receptor sites and two sensitized polynucleotide DNA probes, each having a different sequence complementary to the analyte comprising performing the hereinbelow steps in the order presented:
-
(a) attaching the first DNA probe to the receptor sites; (b) then performing the following two substeps in any order, that is sequentially or in reverse order; (1) hybridizing the oligonucleotide analyte to the complementary portions of the first and second DNA probes; and
,(2) labeling by the attachment of an indicator to the second DNA probe; and
,(c) detecting the intensity of the indicator and thereby obtaining a quantitation of said oligonucleotide analyte. - View Dependent Claims (2, 3, 4, 5, 6, 7)
-
-
8. A method of quantitative analysis of a oligonucleotide analyte, said analysis using an assay plate prepared with masked receptor sites, a polynucleotide DNA probe-for-ligation, and a DNA probe-for-detection comprising performing the hereinbelow steps in the order presented:
-
(a) reacting the DNA probe-for-ligation for the binding thereof, said ligating DNA probe being complementary to a first portion of the oligonucleotide analyte and, upon reaction thereof, being bindable to the receptor site; (b) attaching the reacted DNA probe-for-ligation, hereinafter the ligating DNA probe to the receptor sites; (c) hybridizing the oligonucleotide analyte to the complementary portion of the attached ligating DNA probe; (d) labeling said DNA probe-for-detection by attaching an enzyme thereto thereby forming an enzyme conjugate, said DNA probe hereinafter said detecting DNA probe, detecting DNA probe-for-detection, being complementary to a second portion of the oligonucleotide analyte; (e) hybridizing the oligonucleotide analyte to the complementary portion of the detecting DNA probe; (f) adding a substrate to react with the enzyme conjugate of the detecting DNA probe and then a reagent to arrest the enzymatic reaction at a preselected time point; (g) detecting the concentration of the enzymatic reaction products at the desired end point; and
,(h) calibrating the detected concentration to indicate the quantity of oligonucleotide analyte present. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16)
-
Specification