Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

Abstract

The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence difference.

What is claimed: 
     
         1 . A kit for identifying a plurality of polymorphic loci comprising:
 a plurality of oligonucleotide probe sets, each probe set comprising (i) a first allele-specific oligonucleotide probe comprising a first target allele-specific portion and a first 5′ upstream primer-specific portion, (ii) a second allele-specific oligonucleotide probe comprising a second target allele-specific portion and a second 5′ upstream primer-specific portion, and (iii) a common locus-specific oligonucleotide probe comprising a target specific portion and a 3′ downstream primer-specific portion;   a ligase; and   an oligonucleotide primer set comprising (i) a first upstream primer containing the same sequence as the first 5′ upstream primer-specific portion of the first allele-specific oligonucleotide probe, (ii) a second upstream primer containing the same sequence as the second 5′ upstream primer-specific portion of the second allele-specific oligonucleotide probe, and (iii) a downstream primer complementary to the 3′ downstream primer-specific portion of the common locus-specific oligonucleotide probe, wherein the first and the second upstream primers each comprise a different fluorescent label.   
     
     
         2 . The kit according to  claim 1 , wherein the ligase is selected from the group consisting of  Thermus aquaticus  ligase,  Thermus thermophilus  ligase,  E. coli  ligase, T4 ligase, and  Pyrococcus  ligase. 
     
     
         3 . The kit according to  claim 1 , wherein the target-allele specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85° C. 
     
     
         4 . The kit according to  claim 1 , wherein the target allele-specific portions of the oligonucleotide probe sets are 20-28 nucleotides long. 
     
     
         5 . The kit according to  claim 1 , wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof. 
     
     
         6 . The kit according to  claim 1 , wherein the oligonucleotide primer sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof. 
     
     
         7 . The kit according to  claim 1 , wherein the oligonucleotide probe sets each comprise a different addressable array-specific portion on one of oligonucleotide probes. 
     
     
         8 . The kit according to  claim 7  further comprising:
 a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. 
 
     
     
         9 . The kit according to  claim 8 , wherein the capture oligonucleotides are selected from the group consisting of ribonucleotides and deoxyribonucleotides. 
     
     
         10 . The kit of  claim 1 , wherein the common locus-specific oligonucleotide probe of each oligonucleotide probe set comprises a different addressable array-specific portion. 
     
     
         11 . The kit according to  claim 10  further comprising:
 a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. 
 
     
     
         12 . The kit according to  claim 11 , wherein the capture oligonucleotides are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, peptide nucleotide analogues, modified peptide nucleotide analogues, and mixtures thereof. 
     
     
         13 . The kit according to  claim 1 , wherein each oligonucleotide primer set comprises a different addressable array-specific portion on one of the oligonucleotide primers. 
     
     
         14 . The kit according to  claim 13  further comprising:
 a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. 
 
     
     
         15 . The kit according to  claim 14 , wherein the capture oligonucleotides are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, peptide nucleotide analogues, modified peptide nucleotide analogues, and mixtures thereof. 
     
     
         16 . The kit according  claim 1  further comprising:
 uracil N-glycosylase. 
 
     
     
         17 . The kit according to  claim 1 , wherein the first target allele-specific portion and the second target allele-specific portion of each oligonucleotide probe sets have an identical nucleotide sequence except for the nucleotide at the 3′ terminus of the allele-specific portion, and wherein the 3′ terminus of the allele-specific portion is the 3′ terminus of the oligonucleotide probe. 
     
     
         18 . The kit according to  claim 17 , wherein, for each probe set of the plurality of probe sets, the 5′ upstream primer-specific portions have an identical nucleotide sequence and the 3′ downstream primer-specific portions have an identical nucleotide sequence. 
     
     
         19 . The kit according to  claim 1 , wherein, for each probe set of the plurality of probe sets, the 5′ upstream primer-specific portions have an identical nucleotide sequence and the 3′ downstream primer-specific portion have an identical nucleotide sequence. 
     
     
         20 . A composition comprising two or more oligonucleotide probe sets for identifying two or more of a plurality of target polymorphic loci, each probe set comprising:
 (i) a first allele-specific oligonucleotide probe comprising a first target allele-specific portion and a first 5′ upstream primer-specific portion, (ii) a second allele-specific oligonucleotide probe comprising a second target allele-specific portion and a second 5′ upstream primer-specific portion, and (iii) a common locus-specific oligonucleotide probe comprising a target specific portion and a 3′ downstream primer-specific portion.   
     
     
         21 . The compositions according to  claim 20 , wherein the target-allele specific portions of the oligonucleotide probes each have a hybridization temperature of 50-85° C. 
     
     
         22 . The composition according to  claim 20 , wherein the target allele-specific portions of the oligonucleotide probe sets are 20-28 nucleotides long. 
     
     
         23 . The composition according to  claim 20 , wherein the oligonucleotide probe sets are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, and mixtures thereof. 
     
     
         24 . The composition according to  claim 20 , wherein the oligonucleotide probe sets comprise an addressable array-specific portion on one of oligonucleotide probes. 
     
     
         25 . The composition according to  claim 24  further comprising:
 a solid support with different capture oligonucleotides immobilized at different particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions. 
 
     
     
         26 . The composition according to  claim 25 , wherein the capture oligonucleotides are selected from the group consisting of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar backbone oligonucleotides, nucleotide analogues, peptide nucleotide analogues, modified peptide nucleotide analogues, and mixtures thereof.