Microparticle containing nucleic acid capture probes and method of use

Abstract

A method of identifying alleles of polymorphic sites in a plurality of nucleic acid samples including the steps of determining a source tag sharing number “d” for each of the alleles; performing a first reaction in a plurality of pools of the alleles to be identified to produce reaction products including a source tag identifying said each pool; pooling the pools to provide pooled pools; for each of the alleles to be identified, performing a second reaction using said reaction products to produce allele-specific second reaction products comprising a marker tag and a derived source tag; identifying said allele-specific second reaction products to identify the alleles. If “d” is equal to or larger than a maximum pool size, the first reaction may not be performed. Alleles may be binned together. A microparticle comprising one or more capture probes each comprising an oligonucleotide complementary to a subsequence of a target polynucleotide.

1 . (canceled) 
     
     
         2 . A microparticle comprising:
 a first capture probe attached to the microparticle by its 5′ terminus and extending from the microparticle, the first capture probe comprising a nucleotide sequence capable of hybridizing to a first subsequence of a target polynucleotide, or to a sequence derived from said first subsequence;   a second capture probe attached to the microparticle by its 3′ terminus and extending from the microparticle, the second capture probe comprising a nucleotide sequence capable of hybridizing to a second subsequence of the target polynucleotide, or to a sequence derived from said second subsequence;   whereby the microparticle is capable of binding the target polynucleotide by hybridization of the first and second capture probes to the first and second subsequences of the target polynucleotide.   
     
     
         3 . The microparticle according to  claim 2  wherein at least one of the first and second capture probes comprises a nucleotide sequence capable of hybridization to a barcode in the first or second subsequence of the target polynucleotide. 
     
     
         4 . The microparticle according to  claim 3  wherein the first capture probe comprises a nucleotide sequence capable of hybridization to a barcode in the first subsequence of the target polynucleotide, and the second capture probe comprises a nucleotide sequence capable of hybridization to a barcode in the second subsequence of the target polynucleotide. 
     
     
         5 . The microparticle according to  claim 2  further comprising an attached tag or label. 
     
     
         6 . The microparticle according to  claim 5 , wherein the attached label comprises a fluorescent label. 
     
     
         7 . The microparticle according to  claim 6 , wherein the fluorescent label is an encoding label. 
     
     
         8 . A microparticle comprising:
 a capture probe attached to the microparticle and extending from the microparticle, the capture probe comprising:
 a first segment capable of hybridizing to a first subsequence of a target polynucleotide, or to a sequence derived from said first subsequence; and 
 a second segment capable of hybridizing to a second subsequence of the target polynucleotide, or to a sequence derived from said second subsequence; 
   whereby the microparticle is capable of binding the target polynucleotide by hybridization of the first and second segments of the capture probe to the first and second subsequences of the target polynucleotide.   
     
     
         9 . The microparticle according to  claim 8  wherein at least one of the first and second capture probes comprises a nucleotide sequence capable of hybridization to a barcode in the first or second subsequences of the target polynucleotide. 
     
     
         10 . The microparticle according to  claim 9  wherein the first capture probe comprises a nucleotide sequence capable of hybridization to a barcode in the first subsequence of the target polynucleotide, and the second capture probe comprises a nucleotide sequence capable of hybridization to a barcode in the second subsequence of the target polynucleotide. 
     
     
         11 . The microparticle according to  claim 8  further comprising an attached tag or label. 
     
     
         12 . The microparticle according to  claim 11 , wherein the attached label comprises a fluorescent label. 
     
     
         13 . The microparticle according to  claim 12 , wherein the fluorescent label is an encoding label. 
     
     
         14 . A method of detecting a polynucleotide comprising first and second target nucleotide subsequences comprising:
 contacting the polynucleotide with a microparticle comprising:
 a first capture probe attached to the microparticle and extending from the microparticle, the first capture probe comprising a nucleotide sequence capable of hybridizing to the first target subsequence of the polynucleotide, or to a sequence derived from said first target subsequence; 
 a second capture probe attached to the microparticle and extending from the microparticle, the second capture probe comprising a nucleotide sequence capable of hybridizing to the second target subsequence of the polynucleotide, or to a sequence derived from said second target subsequence; and 
   detecting the polynucleotide upon capture of the polynucleotide by the microparticle.   
     
     
         15 . The method of  claim 14  wherein the polynucleotide is detected by detecting hybridization thereof to the first and second capture probes. 
     
     
         16 . The method according to  claim 14  wherein the first capture probe is attached to the microparticle by its 5′ terminus, and the second capture probe attached to the microparticle by its 3′ terminus. 
     
     
         17 . The method according to  claim 14  wherein at least one of the first and second target subsequences of the polynucleotide comprises a barcode. 
     
     
         18 . The method according to  claim 17  wherein both the first and second target subsequences of the polynucleotide comprise barcodes. 
     
     
         19 . The method according to  claim 14  wherein the microparticle comprises an attached tag or label. 
     
     
         20 . The method according to  claim 19 , wherein the attached label comprises a fluorescent label. 
     
     
         21 . The microparticle according to  claim 20 , wherein the fluorescent label is an encoding label. 
     
     
         22 . The method according to  claim 14 , wherein the polynucleotide comprises a label. 
     
     
         23 . The method according to  claim 22 , wherein label comprises an optical label.