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 primary oligonucleotide primer sets, each primer set comprising (i) a first oligonucleotide primer comprising a target-specific portion and a 5′ upstream secondary primer-specific portion and (ii) a second oligonucleotide primer comprising a target-specific portion and a 5′ upstream, secondary primer-specific portion;   a polymerase; and   an oligonucleotide secondary primer set comprising (i) a first secondary primer containing the same sequence as the 5′ upstream primer-specific portion of the first primary oligonucleotide primer and (ii) a secondary primer containing the same sequence as the 5′ upstream portion of the second primary oligonucleotide primer, wherein the first or second secondary primers comprise a detectable label.   
     
     
         2 . 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. 
     
     
         3 . The kit according to  claim 1 , wherein the oligonucleotide primer sets comprise an addressable array-specific portion on one of the oligonucleotide primers. 
     
     
         4 . The kit according to  claim 3  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. 
 
     
     
         5 . The kit according to  claim 4 , 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. 
     
     
         6 . The kit according to  claim 1 , wherein the detectable label is a fluorescent label. 
     
     
         7 . A composition comprising a plurality of primary oligonucleotide primer sets, each primer set comprising:
 (i) a first oligonucleotide primer comprising a target-specific portion and a 5′ upstream secondary primer-specific portion and (ii) a second oligonucleotide primer comprising a target-specific portion and a 5′ upstream secondary primer-specific portion.   
     
     
         8 . The composition according to  claim 7 , 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. 
     
     
         9 . The composition according to  claim 7 , wherein the oligonucleotide primer sets comprise an addressable array-specific portion on one of the oligonucleotide primers. 
     
     
         10 . The composition according to  claim 9  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. 
 
     
     
         11 . The composition according to  claim 10 , 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.