Immobilized oligonucleotide probes and uses therefor

Abstract

The invention is a method of detecting nucleic acids in a sample using oligonucleotide probes which are noncovalently bound to solid supports for rapid, sensitive, hybridization assays. The method involves coating the support surface with a polynucleotide and then hybridizing a specific capture probe for each analyte to the polynucleotide by way of a short tail of the complementary polynucleotide. The immobilized probes are used to capture nucleic acid targets out of complex specimens for nonisotopic detection without the need for prior cell culture or purification of the target nucleic acids. A panel of tests can be run on each specimen simultaneously, a format that conserves precious samples. The assay can be readily automated, and can be conveniently run in a manual fashion on large numbers of samples in two to three hours.

1 . A method of isolating or determining a target nucleic acid in a sample comprising contacting the sample with a solid support coated with a substratum hybridized to a capture probe which is complementary to the target nucleic acid, under conditions appropriate for the target nucleic acid to hybridize with the capture probe.  
     
     
         2 . A method of  claim 1 , wherein the solid support comprises polystyrene.  
     
     
         3 . A method of  claim 2 , wherein the solid support is selected from the group consisting of: 
 polystyrene microtiter wells, polystyrene tubes and polystyrene dipsticks.    
     
     
         4 . A method of  claim 1 , wherein the substratum comprises a polynucleotide.  
     
     
         5 . A method of  claim 4 , wherein the substratum is poly(dT) or poly(dA).  
     
     
         6 . A method of  claim 1 , wherein the prehybridized oligonucleotide probe comprises an oligonucleotide linked to a homopolynucleotide tail section which tail section is complementary to the substratum.  
     
     
         7 . A method of  claim 6 , wherein the substratum comprises poly(dT) and the tail section comprises poly(dA).  
     
     
         8 . A method of  claim 6 , wherein the substratum comprises poly(dA) and the tail section comprises poly(dT).  
     
     
         9 . A method of  claim 1 , wherein said tail is attached to the 3′ end or 5′ end, or is bound to at least one internal residue of the oligonucleotide probe.  
     
     
         10 . A method of  claim 1 , wherein said oligonucleotide probe is DNA and said tail is DNA or RNA.  
     
     
         11 . A method of  claim 1 , wherein said oligonucleotide probe is RNA and said tail is DNA or RNA.  
     
     
         12 . A method of  claim 1 , wherein said tail is a homopolynucleotide sequence or a mixed base sequence of two or more nucleotides.  
     
     
         13 . A method of  claim 1 , wherein the substratum is multilayered.  
     
     
         14 . A method of determining a target nucleic acid in a specimen comprising the steps of: 
 a. contacting the specimen with an agent that disrupts the molecular structure;    b. contacting the mixture obtained in (a) with a probe comprising a solid support having coated thereon a substratum to which an oligonucleotide complementary to the target nucleic acid is prehybridized, under conditions appropriate for the target nucleic acid to hybridize with the oligonucleotide probe;    c. labeling the hybridized target nucleic acids; and    d. detecting the labeled targets.    
     
     
         15 . A method of  claim 14 , wherein the solid support is comprised of polystyrene.  
     
     
         16 . A method of  claim 15 , wherein the solid support comprises polystyrene microtiter wells polystyrene tubes or polystyrene dipsticks.  
     
     
         17 . A method of  claim 14 , wherein the substratum comprises a polynucleotide.  
     
     
         18 . A method of  claim 17 , wherein the substratum comprises poly(dT) or poly(dA).  
     
     
         19 . A method of  claim 14 , wherein the prehybridized oligonucleotide probe comprises an oligonucleotide linked to a polynucleotide tail section which tail section is complementary to the substratum.  
     
     
         20 . A method of  claim 19 , wherein the substratum comprises poly(dT) and the tail section comprises poly(dA).  
     
     
         21 . A method of  claim 19 , wherein the substratum comprises poly(dA) and the tail section comprises poly(dT).  
     
     
         22 . A method of  claim 14 , wherein the method further comprises washing the probe after step 6 with a tetraalkylammonium salt.  
     
     
         23 . A method of  claim 22 , wherein the tetraalkylammonium salt is selected from the group consisting of: tetraethylammonium chloride, tetraethylammonium bromide tetramethylammonium chloride and tetramethylammonium bromide.  
     
     
         24 . A method of  claim 14 , wherein the nucleic acid comprises nucleic acids derived from bacteria or viruses.  
     
     
         25 . A method of reducing nonspecific binding of a labeled probe to the support having a target nucleic acid bound thereto, and of increasing the size of the signal, the method comprising washing said solid supports with a wash buffer appropriate to lower nonspecific binding wherein said buffer is applied after the capture of a target nucleic acid but before the addition of the labeled probe.  
     
     
         26 . The method  claim 23  comprising washing solid supports to which target molecules are bound by means of one of the following affinity pairs: oligo(dT)-poly(dA), oligo(dA)-poly(dT), oligo(dT)-oligo(dA), or poly(dT)-poly(dA).  
     
     
         27 . The method of  claim 26  comprising employing a set of washing conditions in which said affinity pair is much stronger than the hybrid pairs involved in a mixed base sequence, such that nonspecific hybridization of the capture probe, or labeled probe to a pseudo-target can be substantially reduced without causing elution of the target nucleic acid from the solid support.  
     
     
         28 . The method of  claim 27 , wherein said wash buffer is a tetraalkylammonium salt.  
     
     
         29 . The method of  claim 27 , wherein said washing conditions refers to the use of a wash buffer that is or contains a tetraalkylammonium salt at a temperature between approximately one and five degrees below the dissociation temperature of the probe that possesses the lowest dissociation temperature with the target.  
     
     
         30 . The method of  claim 27 , wherein the signal is increased due to enhanced binding or enhanced hybridization of the labeled probe to the target.  
     
     
         31 . A method of removing the effect of potential interfering substances from a sample prior to the addition of a labeled probe to the sample, comprising the steps of: 
 a. solubilizing the nucleic acids of each sample with a chaotropic solvent;    b. contacting the sample with solid supports to which oligonucleotides specific for the target nucleic acids are pre-immobilized thereby capturing the target nucleic acids;    c. labeling the captured target nucleic acids with a labeled probe; and    d. detecting the labeled target nucleic acids.    
     
     
         32 . The method of  claim 31  in which said interfering substances that are removed in step (b) are molecules selected from the group consisting of: endogenous biotin, endogenous avidin, endogenous biotin-binding activities, nucleic acid binding molecules, molecules that can bind the labeled probe, proteases and nucleases.  
     
     
         33 . A method of determining the presence or amount of target nucleic acid in a sample using a reversible target capture protocol comprising the steps of: 
 a. providing a first capture probe comprising an oligonucleotide preimmobilized to a first substratum through a short tail;    b. contacting the probe with the sample under hybridizing conditions appropriate to allow hybridization of the target nucleic acid with the bound oligonucleotide and wherein the hybrid between the short tail of the first oligonucleotide probe with the first substratum is stable;    c. eluting the probe under conditions appropriate to destabilize the short tail-first substratum hybrid or the first oligonucleotide probe-target hybrid;    d. adding a second capture probe having a preimmobilized oligonucleotide sequence specific for the target nucleic acid, wherein said oligonucleotide is bound to the support through a long tail;    e. recapturing the target nucleic acid by means of the second capture probe;    f. labeling said target nucleic acid; and    g. measuring said labeled target nucleic acid.    
     
     
         34 . The method of  claim 34 , wherein the short tail is in the range of approximately 10-20 nucleotides and the long tail is greater than about 30 nucleotides.  
     
     
         35 . The method of  claim 33 , wherein the short tail-substratum hybrid is stabilized by adding a solvent appropriate to strengthen the A-T (A-U) base pair relative to the G-C base pair, or to strengthen the poly(dA or rA)-oligo(dT or U) or poly(dT or U)-oligo(dA or rA) or poly(dA or rA)-poly(dT or U) homopolymer relative to mixed base sequences.  
     
     
         36 . The method of  claim 35 , wherein said solvent comprises a tetraalkylammonium salt.  
     
     
         37 . The method of  claim 33 , wherein said elution is performed by contacting the probe with a chaotropic solvent.  
     
     
         38 . The method of  claim 38 , wherein the chaotropic solvent is GuSCN.  
     
     
         39 . The method of  claim 34 , wherein the second capture probe has the same mixed based sequence as the first capture probe.  
     
     
         40 . The method of  claim 34 , wherein the second capture probe has a different mixed base sequence from the first capture probe, thereby enhancing the specificity of the hybridization reaction and the assay.  
     
     
         41 . A method of reversible target capture capable of enhancing the overall specificity of an assay, comprising the steps of: 
 a. providing a first solid support having a first tailed oligonucleotide probe hybridized thereto;    b. contacting a second tailed oligonucleotide probe with a second solid support, said second probe having a different mixed base sequence from said first oligonucleotide probe;    c. contacting the first solid support of (a) with a sample containing target nucleic acids, thereby capturing target nucleic acids on said first solid support;    d. eluting target nucleic acids from the first solid support;    e. contacting the target nucleic acids from (d) the second solid support thereby capturing the target nucleic acids on said second solid support;    f. labeling said target nucleic acids; and    g. detecting said target nucleic acids.    
     
     
         42 . The method of  claim 41 , wherein said first solid support has a short or a long complementary nucleotide sequence attached to it.  
     
     
         43 . The method of  claim 41 , wherein elution of the target from the first solid support also removes the tailed oligonucleotide probe, said first tailed probe being prevented from binding to the second solid support because said second solid support remains substantially saturated with said second capture probe during the entire second capture process.  
     
     
         44 . The method of  claim 41 , wherein a homopolymer complementary to the tail on the first capture probe is added prior to the addition to the sample of the second solid support; said homopolymer being added in a quantity in excess over the amount required to hybridize to all of the tails on said first capture probe, and said homopolymer being incubated with said tail on the first capture probe for an appropriate length of time such that essentially no free tail is available to bind to said second solid support.  
     
     
         45 . The method of  claim 41 , wherein a homopolymer complementary to the homopolymer sequence on the second solid support is incubated with the second support for a sufficient period of time prior to the addition of the sample to said second support to prebind any free sequences on said second support, such that said first capture probe is substantially prevented from binding to said second solid support.  
     
     
         46 . The method of  claim 41 , wherein elution of the target from the first solid support does not remove said first tailed capture probe from said first solid phase because the elution occurs under a set of conditions that stabilizes the substratum-tail hybrid relative to the mixed base sequence hybrid.  
     
     
         47 . The method of  claim 46 , wherein said conditons comprise the use of a tetraalkylammonium salt for said first elution at a temperature in which the mixed base sequence hybrid is unstable while the tail-substratum hybrid is stable.  
     
     
         48 . A kit comprising: 
 a. suitable solid supports, each containing a bound substratum and a specific tailed capture probe prehybridized to the substratum; and    b. an agent that disrupts molecular structures.    
     
     
         49 . A kit of  claim 48 , wherein the solid supports comprise agarose beads, polystyrene microtiter wells, polystyrene tubes or polystyrene dipsticks.  
     
     
         50 . A kit of  claim 48 , wherein the substratum comprises a polynucleotide.  
     
     
         51 . A kit of  claim 48 , wherein the substratum comprises poly(dT) and the capture probe tail comprises poly(dA).  
     
     
         52 . A kit of  claim 48 , wherein the substratum comprises poly(dA) and the capture probe tail comprises poly(dT).  
     
     
         53 . A kit of  claim 48 , further comprising a wash buffer, a labeled probe, a means for detecting the labeled probe, one or more positive control samples, one or more negative control samples, elution buffers for reversible target capture, or amplification or cloning reagents.  
     
     
         54 . A kit comprising: 
 a. suitable solid supports, each having a bound substratum;    b. a separate tailed capture probe, wherein the tail is complementary to the substratum, and    c. an agent that disrupts molecular structures.    
     
     
         55 . A kit comprising: 
 a. suitable solid supports, each containing a bound substratum and a specific capture probe prehybridized to the substratum;    b. lysis solution;    c. labeled probe;    d. wash buffer;    e. a means for detecting said labeled probe;    f. one or more positive control samples;    g. one or more negative control samples;    h. elution buffers for reversible target capture; and    i. amplification or cloning reagents.    
     
     
         56 . A device comprising: 
 a. a pipetting station and a detection apparatus, said pipetting station being capable of performing sequential operations of adding and removing reagents to the solid phases at specified time points in a thermostatted environment;    b. said sequential operations comprising: 
 i. mixing samples, lysis solutions, and solid supports;  
 ii. withdrawing fluid;  
 iii. adding wash buffer;  
 iv. repeating steps two and three several times;  
 v. adding optional stringency wash buffer, or elution buffer;  
 vi. removing optional stringency wash buffer, or elution buffer;  
 vii. adding labeled probe(s);  
 viii. repeating steps two and three several times;  
 ix. adding detection agents;  
 x. repeating steps two and three several times; and  
 xi. detecting signal with said detection apparatus.