Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection

Abstract

The invention provides methods and processes for the identification of polymorphisms at one or more designated sites, without interference from non-designated sites located within proximity of such designated sites. Probes are provided capable of interrogation of such designated sites in order to determine the composition of each such designated site. By the methods of this invention, one or more mutations within the CFTR gene and the HLA gene complex can be can be identified.

We claim  
     
         1 . A method of concurrent determination of nucleotide composition at designated polymorphic sites located within one or more target nucleotide sequences, said method comprising the following steps: 
 (a) providing one or more sets of probes, each probe capable of annealing to a subsequence of said one or more target nucleotide sequences located within a range of proximity to a designated polymorphic site;    (b) contacting the set of probes with said one or more target nucleotide sequences so as to permit formation of hybridization complexes by placing an interrogation site within a probe sequence in direct alignment with the designated polymorphic site;    (c) for each hybridization complex, determining the presence of a match or a mismatch between the interrogation site and a designated polymorphic site; and    (d) determining the composition of the designated polymorphic site.    
     
     
         2 . The method of  claim 1  wherein said one or more target nucleotide sequences are produced in a multiplex PCR reaction using one or more primer sets.  
     
     
         3 . The method of  claim 2  wherein said primers sets are degenerate primer sets.  
     
     
         4 . The method of  claim 1  wherein said targets are fragments of genomic DNA.  
     
     
         5 . The method of  claim 1  wherein said targets are fragments of cDNA.  
     
     
         6 . The method of  claim 1  wherein one or more sets of probes are spatially encoded on a substrate.  
     
     
         7 . The method of  claim 1  wherein one or more sets of probes are immobilized on encoded microparticles.  
     
     
         8 . The method of  claim 7  wherein the encoded microparticles are assembled into a random encoded array.  
     
     
         9 . The method of  claim 1  wherein each probe contains a terminal elongation initiation region capable of initiating an elongation or extension reaction.  
     
     
         10 . The method of  claim 9  wherein the reaction is catalyzed by a polymerase lacking 3′→5′ exonuclease activity.  
     
     
         11 . The method of  claim 1  wherein step (c) comprises adding one or more deoxynucleotide triphosphates.  
     
     
         12 . The method of  claim 11  further comprising using a polymerase capable of extending or elongating probes.  
     
     
         13 . The method of  claim 12  wherein the polymerase lacks 3′→5′ exonuclease activity.  
     
     
         14 . The method of  claim 11  wherein at least one of the deoxy nucleotide triphosphates is labeled so as to generate an optically detectable signature associated with the elongation product.  
     
     
         15 . The method of  claim 1  wherein an optical label is attached to one or more probes by annealing to the probes a fluorescently labeled target to form a fluorescent hybridization complex.  
     
     
         16 . The method of  claim 15  further comprising using a polymerase capable of extending or elongating probes displaying a match by addition of one or more deoxynucleotide triphosphates to form an elongated hybridization complex.  
     
     
         17 . The method of  claim 16  further comprising identifying elongation products by detecting the stability of optical signatures under conditions in which temperature is set to a value above the melting temperature of any hybridization complex formed by target and non-matched probe but below the melting temperature of any extended hybridization complex formed by target and elongated probe.  
     
     
         18 . The method of  claim 15  wherein one or more probes from the set of probes are immobilized on encoded microparticles and a change in optical signature is detected.  
     
     
         19 . The method of  claim 15  wherein one or more probes from the set of probes are immobilized on encoded microparticles which are arranged in random encoded arrays.  
     
     
         20 . The method of  claim 19  wherein the arrays are arranged in a spatially encoded manner.  
     
     
         21 . The method of  claim 15  wherein the change in optical signature is detected and particle identity is determined.  
     
     
         22 . A method of sequence-specific amplification of assay signals produced in the analysis of a nucleic acid sequence of interest in a biological sample, comprising the following steps: 
 (a) providing a set of immobilized probes capable of forming a hybridization complex with the sequence of interest;    (b) contacting said set of immobilized probes with said biological sample containing said sequence of interest under conditions which permit the sequence of interest to anneal to at least one of the immobilized probes to form a hybridization complex;    (c) contacting said hybridization complex with a polymerase to allow elongation or extension of the probes contained within said hybridization complex;    (d) converting elongation or extension of the probes into an optical signal; and    (e) recording said optical signal from the set of immobilized probes in real time.    
     
     
         23 . The method of  claim 22  further comprising performing one or more cycles, each cycle comprising “annealing-extending/elongating-detecting-denaturing” steps, wherein each cycle results in the increase of the number of extended or elongated probes in arithmetic progression.  
     
     
         24 . The method of  claim 23  comprising the steps of: 
 (a) setting a first temperature favoring the formation of a hybridization complex;  
 (b) setting a second temperature favorable to polymerase-catalyzed extension;  
 (c) converting extension or elongation into optical signal;  
 (d) recording/imaging optical signals/signatures from all immobilized probes; and  
 (e) setting a third temperature so as to ensure denaturation of all hybridization complexes.  
 
     
     
         25 . A method of forming a covering probe set for the concurrent interrogation of a designated polymorphic site located in one or more target nucleic acid sequences comprising the steps of: 
 (a) determining the sequence of an elongation probe capable of alignment of the interrogation site of the probe with a designated polymorphic site;    (b) further determining a complete set of degenerate probes to accommodate all non-designated as well as non-selected designated polymorphic sites while maintaining alignment of the interrogation site of the probe with the designated polymorphic site; and    (c) reducing the degree of degeneracy by removing all tolerated polymorphisms.    
     
     
         26 . The method of  claim 25  wherein the covering set contains at least two probes with different interrogation site composition per designated site.  
     
     
         27 . The method of  claim 25  wherein the reduction of complexity in step (c) is accomplished by probe pooling.  
     
     
         28 . A method of identifying polymorphisms at one or more designated sites on one or more target nucleotides, the method comprising 
 (a) providing one or more probes capable of interrogating said designated sites;    (b) forming an elongation product by elongating one or more probes designed to interrogate a designated site; and    (c) determining the compositions at said two or more sites.    
     
     
         29 . The method of  claim 28  further comprising forming a hybridization complex by annealing to the elongation product a second probe designed to interrogate a second designated site.  
     
     
         30 . A method for identifying polymorphisms at one or more designated sites within a target polynucleotide sequence, the method comprising 
 (a) providing one or more probes capable of interrogating said designated sites;    (b) assigning a value to each such designated site while accommodating non-designated polymorphic sites located within a range of proximity to each such polymorphism.    
     
     
         31 . The method of  claim 30  wherein the homology between the probes and the target sequence is analyzed by multiplexing.  
     
     
         32 . A method for determining polymorphism at one or more designated sites of a target nucleotide sequence, the method comprising the steps of providing one or more pairs of probes capable of detecting deletions wherein the deletions are placed either at the 3′ terminus of the probe or within 3-5 bases of the 3′ terminus.  
     
     
         33 . A method of identifying polymorphisms at two or more designated sites of a target nucleotide sequence, the method comprising 
 (a) selecting a multiplicity of designated polymorphic sites to permit allele assignment;    (b) providing two or more probes capable of concurrent interrogation of the multiplicity of designated sites;    (c) assigning a value to each such designated site; and    (d) combining said values to determine the identity of an allele or group of alleles while accommodating non-designated sites near said designated polymorphisms.    
     
     
         34 . A method for determining a polymorphism at one or more designated sites in a target polynucleotide sequence, the method comprising providing a probe set for such designated sites and grouping said probe set in different probe subsets according to the terminal elongation initiation of each probe.  
     
     
         35 . The method of  claim 34  further comprising the step of multiplexing said probe set, measuring each probe in the probe set without interference from the other probes in the probe set and changing the allele matching pattern of a target polynucleotide sequence to include alleles that are tolerated by a probe set.  
     
     
         36 . The method of  claim 35  wherein the step of changing the allele matching pattern of a target polynucleotide sequence comprises pooling one or more probe sets to include matched alleles.  
     
     
         37 . The method of  claim 36  wherein the step of changing the allele matching pattern of a target polynucleotide sequence comprises the step of comparing the signal intensities produced by the probe set.  
     
     
         38 . The method of  claim 37  further comprising the step of separating the terminal elongation initiation region and duplex anchoring region on the probe set.  
     
     
         39 . A method for the concurrent interrogation of a multiplicity of polymorphic sites comprising the step of conducting a multiplexed elongation assay by applying one or more temperature cycles to achieve linear amplification of such target.  
     
     
         40 . A method for the concurrent interrogation of a multiplicity of polymorphic sites comprising the step of conducting a multiplexed elongation assay by applying a combination of annealing and elongation steps under temperature-controlled conditions.  
     
     
         41 . A method of concurrent interrogation of nucleotide composition at S polymorphic sites, P sub S:={c sub P (s); 1<=s<=S} located within one or more contiguous target sequences, said method assigning to each c sub P one of a limited set of possible values by performing the following steps: 
 (a) providing a set of designated immobilized oligonucleotide probes, also known as elongation probes, each probe capable of annealing in a preferred alignment to a subsequence of the target located proximal to a designated polymorphic site, the preferred alignment placing an interrogation site within the probe sequence in direct juxtaposition to the designated polymorphic site, the probes further containing a terminal elongation initiation (TEI) region capable of initiating an elongation or extension reaction;    (b) permitting the one or more target sequences to anneal to the set of immobilized oligonucleotide probes so as form probe-target hyrbdization complexes; and    (c) for each probe-target hybridization complex, calling a match or a mismatch in composition between interrogation site and corresponding designated polymorphic site.    
     
     
         42 . The method of  claim 41 , wherein probes are immobilized in a spatially encoded fashion on a substrate.  
     
     
         43 . The method of 41, wherein probes are immobilized on encoded microparticles which are in turn assembled in a random encoded array on a substrate.  
     
     
         44 . The method of 41, in which the calling step involves the use of a polymerase capable of extending or elongating probes whose interrogation site composition matches that of the designated polymorphic site in the target, and only those probes, by addition of one or more nucleoside triphosphates, one of which is labeled so as to generate an optically deectable signature  
     
     
         45 . The method of  claim 41 , wherein an optical signature is attached to all available immobilized probes in the first step by annealing to these primers a fluorescently labeled target to form a fluorescent hybridization complex, and wherein the second step involves the use of a polymerase capable of extending or elongating probes displaying a terminal match, and only those probes, by addition of one or more nucleotide triphosphates to form an extended hybridization complex, and wherein extension products are identified by the stability of optical signatures under an increase in temperature to a value selected to exceed the melting temperature of any hybridization complex but not to exceed the melting temperature of any extended hybridization complex.  
     
     
         46 . The method of  claim 45 , wherein probes are immobilized on encoded microparticles and the change in optical signature is detected, and particle identity determined, by flow cytometry.  
     
     
         47 . The method of  claim 45 , wherein probes are immobilized on encoded microparticles which are arranged in random encoded arrays, said arrays optionally arranged in a spatially encoded manner, and the change in optical signature is detected, and particle identity is determined, by direct imaging.  
     
     
         48 . A method of sequence-specific amplification of assay signals produced in the analysis of a nucleic acid sequence of interest in a biological sample, the method permitting real-time monitoring of amplified signal, and comprising the following steps: 
 (a) providing a temperature-controlled sample containment device with associated temperature control apparatus permitting real-time recording of optical assay signal produced within said device;    (b) providing within said sample containment device a set of distinguishable, immobilized oligonucleotide probes capable forming a hybridization complex with the sequence of interest;    (c) permitting the sequence to anneal to the set of immobilized oligonucleotide probes to form a hybridization complex;    (d) contacting said hybridization complex with a polymerase to allow elongation of extension of the matched probes contained within a hybridization complex;    (e) providing means to convert elongation or extension of matching probes into an optical assay signal;    (f) providing an optical recording/imaging device capable of recording optical assay signals from the set of immobilized probes in real time;    (g) performing one or more “annealing-extending-detecting-denaturing” cycles, each cycle increasing the number of extended or elongated probes in arithmetic progression and involving the following steps: 
 (i) set a first temperature favoring the formation of a hybridization complex;  
 (ii) set a second temperature favorable to polymerase-catalyzed extension;  
 (iii) convert extension into optical signal;  
 (iv) record/image optical signals/signatures from all immobilized probes; and  
 (v) set a third temperature so as to ensure denaturation of all hybridization complexes.