Methods, kits and compositions for supressing the binding of detectable probes to non-target sequences in hybridization assays

Abstract

This invention relates to methods, kits and compositions suitable for the improved detection, analysis and quantitation of nucleic acid target sequences using probe based hybridization assays. The invention is more specifically directed to methods, kits and compositions suitable for suppressing the binding of detectable nucleic acid probes or detectable PNA probes to non-target nucleic acid sequences in an assay for a target nucleic acid sequence to thereby improve the reliability, sensitivity and specificity of the assay. The methods, kits and compositions of this invention are particularly well suited to the detection and analysis of nucleic acid point mutations.

We claim:  
     
         1 . A method for suppressing the binding of a detectable probe to a non-target sequence in an assay of a sample for a target sequence, the method comprising the steps of: 
 a. contacting the sample with a set containing two or more probes under conditions suitable for the probes to hybridize to nucleic acid, wherein, at least one of said probes is a detectable probe labeled with a detectable moiety and having a sequence complementary or substantially complementary to the target sequence, and at least one of the other probes is an unlabeled or independently detectable probe having a sequence complementary or substantially complementary to a non-target sequence which may be present in the sample; provided that at least one of the detectable probe and the unlabeled or independently detectable probe is a PNA probe; and    b. detecting the presence or amount of target sequence present in the sample by directly or indirectly detecting or quantitating the detectable moiety of said detectable probe which hybridized to the target sequence.    
     
     
         2 . The method of  claim 1 , wherein all probes of the set are PNA probes.  
     
     
         3 . The method of  claim 1 , wherein the target sequence is DNA or RNA.  
     
     
         4 . The method of  claim 1 , wherein the target sequence is immobilized to a surface.  
     
     
         5 . The method of  claim 4 , further comprising the step of: 
 c. collecting an aliquot, by elution from the surface, containing any of the one or more detectable probes which hybridized to target sequence when performing step (a.).    
     
     
         6 . The method of  claim 5 , wherein each of the detectable probes, having a distinct sequence, comprise independently detectable moieties which are used to identify or quantitate the presence or amount of each distinct probe sequence present in the aliquot collected.  
     
     
         7 . The method of  claim 6 , wherein the detectable moiety of the detectable probe is a mass marker and the identity of each distinct probe in the aliquot is determined by mass analysis using Positive-ion Fast Atom Bombardment Tandem Mass Spectrometry.  
     
     
         8 . The method of  claim 1 , wherein target and non-target sequences are closely related sequences.  
     
     
         9 . The method of  claim 8 , wherein target sequence and the non-target sequence are related as a point mutation.  
     
     
         10 . The method of  claim 1 , wherein the assay is used to detect, identify, or quantitate the presence or amount of an organism or virus in the sample.  
     
     
         11 . The method of  claim 1 , wherein the assay is used to detect, identify, or quantitate the presence or amount of one or more species of an organism in the sample.  
     
     
         12 . The method of  claim 1 , wherein the assay is used to determine the effect of antimicrobial agents on the growth of one or more microorganisms in the sample.  
     
     
         13 . The method of  claim 1 , wherein the assay is used to determine the presence or amount of a taxonomic group of organisms in the sample.  
     
     
         14 . The method of  claim 1 , wherein each of the different detectable PNA probes of the set comprise independently detectable moieties.  
     
     
         15 . The method of  claim 1 , wherein the detectable moiety is selected from the group consisting of a chromophore, a fluorochrome, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.  
     
     
         16 . The method of  claim 15 , wherein the enzyme is selected from the group consisting of alkaline phosphatase, soybean peroxidase and horseradish peroxidase.  
     
     
         17 . The method of  claim 15 , wherein the hapten is selected from the group consisting of fluorescein, biotin, 2,4-dinitrophenyl and digoxigenin.  
     
     
         18 . The method of  claim 1 , wherein step (a.) comprises 
 i.) incubating the sample with at least one unlabeled or independently detectable probe having a sequence complementary or substantially complementary to a non-target sequence which may be present in the sample for a first period of time under conditions suitable for probes to hybridize to nucleic acid; and    ii.) incubating the sample with the at least one detectable probe labeled with a detectable moiety and having a sequence complementary or substantially complementary to a target sequence for a second period of time, under conditions suitable for probes to hybridize to nucleic acid.    
     
     
         19 . The method of  claim 18 , wherein; 
 i.) each of the first period of time and the second period or time is twenty minutes or less;    ii.) at least one detectable probe is a PNA probe; and    iii.) at least one unlabeled or independently detectable probe is a PNA probe.    
     
     
         20 . The method of  claim 1 , wherein the ratio of unlabeled or independently detectable probe to detectable probe is at least two to one  
     
     
         21 . A kit suitable for suppressing the binding of a detectable probe to a non-target sequence in an assay of a sample for a target sequence, said kit comprising, a set of two or more probes wherein, at least one of the probes is a detectable probe labeled with a detectable moiety and having a sequence complementary or substantially complementary to a target sequence, and at least one of the other probes is an unlabeled or independently detectable probe having a sequence complementary or substantially complementary to a non-target sequence which may be present in the sample; provided that at least one of the detectable probe and the unlabeled or independently detectable probe is a PNA probe.  
     
     
         22 . The kit of  claim 21 , wherein all probes of the set are PNA probes.  
     
     
         23 . The kit of  claim 21 , wherein each of the different detectable probes of the set comprise independently detectable moieties.  
     
     
         24 . The kit of  claim 21 , wherein each of the different detectable probes of the set are nucleic acid probes and all other probes of the set are unlabeled PNA probes each having a defined sequence which is complementary to a non-target sequence which may be present in the sample.  
     
     
         25 . The kit of  claim 22 , wherein the different detectable probes of the set are PNA probes comprising independently detectable moieties.  
     
     
         26 . The kit of  claim 21 , wherein the kit is designed to detect, identify, or quantitate the presence or amount of an organism or virus in the sample.  
     
     
         27 . The kit of  claim 21 , wherein the kit is designed to detect, identify, or quantitate the presence or amount of one or more species of an organism in the sample.  
     
     
         28 . The kit of  claim 21 , wherein the kit is used to determine the effect of antimicrobial agents on the growth of one or more microorganisms in the sample.  
     
     
         29 . The kit of  claim 22  comprising two PNA probes, wherein the first PNA probe has a sequence complementary to the target sequence and is labeled with a detectable moiety and the second PNA probe is unlabeled and has a sequence complementary to a non-target sequence which may be present in the sample.  
     
     
         30 . The kit of  claim 29 , wherein each of the two PNA probes are designed to hybridize specifically to complementary target and non-target sequences wherein the target and non-target sequences are related as point mutations.  
     
     
         31 . The kit of  claim 21  comprising a set of four probes, wherein the probes of the set consist of a single detectable probe labeled with a detectable moiety and having a sequence complementary to the target sequence, and three unlabeled probes each having a defined sequence which is complementary to a non-target sequence which may be present in the sample.  
     
     
         32 . The kit of  claim 31 , wherein the probes of the kit are PNA probes which are designed to hybridize to target and non-target sequences which are related as point mutations.  
     
     
         33 . The kit of  claim 31 , wherein the detectable probe is a nucleic acid probe and the other probes of the kit are unlabeled PNA probes each having a defined sequence which is complementary to a non-target sequence which may be present in the sample.  
     
     
         34 . A composition for suppressing the binding of a detectable probe to a non-target sequence in an assay of a sample for a target sequence, said composition consisting of a set of two probes, wherein, one of the probes is a detectable probe labeled with a detectable moiety and having a sequence complementary to the target sequence, and the other probe is an unlabeled probe having a sequence complementary to a non-target sequence which may be present in the sample, provided that either of the detectable probe or the unlabeled or independently detectable probe is a PNA probe.  
     
     
         35 . The composition of  claim 34 , wherein the probes of the composition are PNA probes which are designed to hybridize specifically to complementary target and non-target sequences wherein the target and non-target sequences are related as point mutations.  
     
     
         36 . The composition of  claim 34 , wherein the detectable probe is a nucleic acid probe and the unlabeled probe is a PNA probe which is complementary to a non-target sequence which may be present in the sample.  
     
     
         37 . A composition for suppressing the binding of a detectable probe to a non-target sequence in an assay of a sample for a target sequence, said composition consisting of a set of four probes wherein, one of the probes is a detectable probe labeled with a detectable moiety and having a sequence complementary to the target sequence, and the other three probes are unlabeled probes each having a defined sequence which is complementary to a non-target sequence which may be present in the sample, provided that at least one of the detectable probe and the unlabeled or independently detectable probe is a PNA probe.  
     
     
         38 . The composition of  claim 37 , wherein the probes of the composition are PNA probes which are designed to hybridize specifically to complementary target and non-target sequences wherein the target and non-target sequences are related as point mutations.  
     
     
         39 . The composition of  claim 37 , wherein the detectable probe is a nucleic acid probe and the three unlabeled probes are PNA probes each having a defined sequence which is complementary to a non-target sequence which may be present in the sample.  
     
     
         40 . A method for suppressing the binding of a non-target sequence to a capture probe immobilized on a surface in a capture assay of a sample for a target sequence of a nucleic acid target molecule, the method comprising the steps of: a. contacting the sample with a solution containing one or more blocking probes under conditions suitable for the blocking probes to hybridize to nucleic acid, wherein the blocking probes are complementary or substantially complementary to one or more non-target sequences which may be present in the sample; 
 b. contacting the sample with the capture probe immobilized on a surface under conditions suitable for the target sequence, if present, to hybridize to the capture probe, wherein the capture probe is complementary or substantially complementary to the target sequence and thereby forms a capture probe/target sequence complex; provided that at least one of a blocking probe or a capture probe is a PNA probe; and    c. detecting the presence or amount of nucleic acid target molecule immobilized to the surface.    
     
     
         41 . The method of  claim 40 , wherein the target nucleic acid molecule is DNA or RNA.  
     
     
         42 . The method of  claim 40 , wherein all probes are PNA probes.  
     
     
         43 . The method of  claim 40 , wherein the target and the non-target sequences are related as a point mutation.  
     
     
         44 . The method of  claim 40 , wherein the surface comprises an array of probes, wherein each distinct probe sequence in the array is designed to capture a specific nucleic acid target molecule, the presence or quantity of which is indicative of the presence or quantity of a specific organism virus, fungi or disease state of interest in the sample  
     
     
         45 . The method of  claim 40 , wherein the presence or amount of nucleic acid target molecule immobilized to the surface is detected using a labeled antibody which specifically interacts with the capture probe/target sequence complex which is formed on the surface.  
     
     
         46 . The method of  claim 45 , wherein the labeled antibody is a labeled anti-nucleic acid/nucleic acid antibody which detects the presence of the capture probe/target sequence complex which is formed by hybridization of the capture probe to the target sequence.  
     
     
         47 . The method of  claim 46 , wherein the labeled antibody is labeled with a detectable moiety selected from the group consisting of a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.  
     
     
         48 . The method of  claim 45 , wherein the labeled antibody is a labeled anti-PNA/nucleic acid antibody which detects the presence of the capture probe/target sequence complex which is formed by hybridization of the PNA capture probe to the target sequence.  
     
     
         49 . The method of  claim 48 , wherein the labeled antibody is labeled with a detectable moiety selected from the group consisting of a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.  
     
     
         50 . The method of  claim 49 , wherein the enzyme is selected from the group consisting of alkaline phosphatase, soybean peroxidase and horseradish peroxidase.  
     
     
         51 . The method of  claim 49 , wherein the hapten is selected from the group consisting of fluorescein, biotin, 2,4-dinitrophenyl and digoxigenin.  
     
     
         52 . The method of  claim 40 , wherein the presence or amount of target sequence immobilized to the surface is detected using a detector probe which hybridizes to a second target sequence of the nucleic acid target molecule.  
     
     
         53 . The method of  claim 52 , wherein the method of  claim 1  is used to suppress the binding of detector probe to a non-second target sequence.  
     
     
         54 . The method of  claim 52 , wherein the detector probe is labeled with a detectable moiety selected from the group consisting of a chromophore, a fluorophore, a spin label, a radioisotope, an enzyme, a hapten and a chemiluminescent compound.  
     
     
         55 . The method of  claim 52 , wherein the detector probe is a nucleic acid probe and the presence or quantity of hybridized detector probe is detected using a labeled anti-nucleic acid/nucleic acid antibody.  
     
     
         56 . The method of  claim 52 , wherein the detector probe is a PNA probe and the presence or quantity of hybridized detector probe is detected using a labeled anti-PNA/nucleic acid antibody.  
     
     
         57 . The method of  claim 40 , wherein the capture assay is used to detect, identify, or quantitate the presence or amount of an organism or virus in the sample.  
     
     
         58 . The method of  claim 40 , wherein the assay is used to detect, identify, or quantitate the presence or amount of one or more species of an organism in the sample.  
     
     
         59 . The method of  claim 40 , wherein the assay IS used to determine the effect of antimicrobial agents on the growth of one or more microorganisms in the sample.  
     
     
         60 . The method of  claim 40 , wherein the assay is used to determine the presence or amount of a taxonomic group of organisms in the sample.