Patent ID: 6171794
Filing Date: 2001-01-09
Classification: C07B,C12Q

Abstract:
A method for determining a contribution of a reference polynucleotide to hybridization of polynucleotides in a sample to a test probe that is immobilized on a first solid surface, said method comprising comparing a dissociation curve for said hybridization to a dissociation profile for the reference polynucleotide, wherein:(a) the dissociation curve is provided by a method comprising(i) contacting a sample to the test probe under conditions that allow polynucleotides in the sample to hybridize to the test probe,(ii) repeatedly washing the test probe under conditions such that some fraction of the hybridized polynucleotides dissociates from the test probe, and(iii) measuring hybridization of the polynucleotides in the sample to the test probe after each of a plurality of said washing steps, over a time period wherein a detectable fraction of hybridized polynucleotides dissociates from the test probe; and(b) the dissociation profile is provided by a method comprising(i) contacting the reference polynucleotide to a reference probe that is immobilized on a second solid surface under conditions that allow the reference polynucleotide to hybridize to the reference probe,(ii) repeatedly washing the reference probe under conditions such that some fraction of the hybridized polynucleotides dissociates from the reference probe, and(iii) measuring hybridization of the reference polynucleotide to the reference probe after each of a plurality of said washing steps, over a time period wherein a detectable fraction of the polynucleotide dissociates from the reference probe; andwherein the contribution of the reference polynucleotide to hybridization to the test probe is the contribution of the dissociation profile to the dissociation curve.