Multiplexed Analysis Of Polymorphic Loci By Concurrent Interrogation And Enzyme-Medicated 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 identified.

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 .- 48 . (canceled) 
     
     
         49 . A probe comprising:
 a. an oligonucleotide sequence comprising a 5′ end and a 3′ end;   b. a Terminal Elongation Initiation (TEI) region comprising the last 4 nucleotides at the 3′ end of the sequence; and   c. a Duplex Anchoring (DA) region which forms an annealing complex with a target nucleotide sequence; wherein the sequence comprises one or more nucleotides corresponding to polymorphisms in the target.   
     
     
         50 . The probe of  claim 49 , wherein said TEI region comprises at least one or more mismatches to the target nucleotide sequence. 
     
     
         51 . The probe of  claim 49 , wherein said DA region exactly matches the corresponding target nucleotide sequence. 
     
     
         52 . The probe of  claim 49 , wherein the DA region is 5′ to the nucleotides corresponding to polymorphisms to the target nucleotide sequence. 
     
     
         53 . The probe of  claim 52 , wherein the DA region is 3-5 nucleotides from at least one mismatch to the target sequence. 
     
     
         54 . The probe of  claim 49 , wherein the TEI region comprises one or more mismatches to the target nucleotide sequence. 
     
     
         55 . The probe of  claim 49 , wherein an elongation reaction can be initiated from the TEI region. 
     
     
         56 . The probe of  claim 49 , wherein the DA region is longer than the TEI region. 
     
     
         57 . The probe of  claim 49 , wherein said probe is immobilized on an encoded substrate. 
     
     
         58 . A method of detecting multiple polymorphisms in a target sequence comprising:
 a. adding a probe to a sample comprising the target, wherein said probe comprises:
 i. an oligonucleotide sequence comprising a 5′ end and a 3′ end; 
 ii. a Terminal Elongation Initiation (TEI) region comprising the last 4 nucleotides at the 3′ end of the oligonucleotide sequence; and 
 iii. a Duplex Anchoring (DA) region which forms an annealing complex with a target sequence;
 wherein the oligonucleotide sequence comprises at least two or more nucleotides corresponding to polymorphisms in the target sequence; 
 
   b. incubating the probe and sample under conditions to allow hybridization of the probe to the target sequence;   c. incubating the probe and sample under conditions to allow an elongation reaction to occur; and   d. detecting the elongation products from the reaction to determine if the multiple polymorphisms are present in the sample.   
     
     
         59 . The method of  claim 58 , wherein the TEI region comprises at least one or more mismatches to the target sequence.