Systems and methods for point-of-care amplification and detection of polynucleotides
First Claim
1. A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
- a. providing a coated, solid support comprising (i) a solid support, (ii) a coating layer comprising at least one polycationic polymer, and (iii) a plurality of target-specific probes attached to the coating layer;
b. contacting the coated, solid support with a sample suspected of having a polynucleotide target and a reaction medium comprising helicase, DNA polymerase, target-specific DNA primers, and dNTPs, wherein the reaction medium has a monovalent cation concentration between about 10 mM and about 100 mM;
c. heating the sample and reaction medium to produce isothermally amplified polynucleotide targets that are simultaneously captured by target-specific probes on the coated, solid support;
d. detecting the presence or absence of the polynucleotide target from amplified polynucleotide targets bound to the target-specific probes.
3 Assignments
0 Petitions
Accused Products
Abstract
Composition and methods for amplifying and detecting solution-state polynucleotide targets in a single device are described. In one aspect, a method for a coupled isothermal amplification and detection process utilizes a coated solid support, including a solid substrate, a cationic layer, and a plurality of target-specific probes attached to the coated solid support. Polynucleotide targets in the sample are amplified by an isothermal amplification process involving in situ hybridization onto the coated solid support. The entire process can be carried out with a high degree of specificity under low salt conditions in less than one hour. Further aspects of the present invention include methods for coupled hybridization/detection of polynucleotide targets, coated silicon biosensors optimized for use with the coupled detection systems to provide visual detection of polynucleotide targets under visible light conditions, and kits for practicing in the above described methods.
147 Citations
24 Claims
-
1. A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
-
a. providing a coated, solid support comprising (i) a solid support, (ii) a coating layer comprising at least one polycationic polymer, and (iii) a plurality of target-specific probes attached to the coating layer; b. contacting the coated, solid support with a sample suspected of having a polynucleotide target and a reaction medium comprising helicase, DNA polymerase, target-specific DNA primers, and dNTPs, wherein the reaction medium has a monovalent cation concentration between about 10 mM and about 100 mM; c. heating the sample and reaction medium to produce isothermally amplified polynucleotide targets that are simultaneously captured by target-specific probes on the coated, solid support; d. detecting the presence or absence of the polynucleotide target from amplified polynucleotide targets bound to the target-specific probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
-
Specification