Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
DC CAFCFirst Claim
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1. A method for identifying a target nucleotide sequence comprising:
- forming a ligation product on a target nucleotide sequence in a ligase 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.
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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 ligase 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 differences.
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11 Claims
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1. A method for identifying a target nucleotide sequence comprising:
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forming a ligation product on a target nucleotide sequence in a ligase 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:
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producing one or more ligation products from a reaction mixture, wherein said reaction mixture comprises; a ligase; one or more target nucleotide sequences; and one or more oligonucleotide probe sets, each said probe set including (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence, wherein a ligation product comprising the first and the second target-specific portions is capable of being produced after the first and the second target-specific portions are hybridized to said corresponding target nucleotide sequence, but is not produced when the first and the second target-specific portions are hybridized with one or more mismatches to a nucleotide sequence present in said reaction mixture, wherein each of said one or more ligation products comprises a ligated sequence which includes (1) the first target-specific portion of the first oligonucleotide probe in a corresponding probe set and (2) the second target-specific portion of the second oligonucleotide probe in said corresponding probe set, or complements thereof; subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more amplification products, each amplification product comprising (1) the ligated sequence of a corresponding ligation product from which said amplification product is amplified, (2) a reporter label, and (3) an addressable array-specific portion, wherein said reporter label and said addressable array-specific portion distinguish said amplification product from other amplification products that comprise other ligated sequences or complements thereof; capturing said one or more amplification products to a solid support; and detecting the reporter labels and the identities of the addressable array-specific portions in said captured amplification products to indicate the presence of one or more target nucleotide sequences in said reaction mixture.
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11. A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:
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subjecting a reaction mixture to one or more ligase detection reaction cycles to produce one or more ligation products, wherein said reaction mixture comprises; a ligase; one or more target nucleotide sequences; and one or more oligonucleotide probe sets, each said probe set including (a) a first oligonucleotide probe comprising a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and (b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence, wherein said first and second oligonucleotide probes are ligated together when the first and the second target-specific portions are hybridized adjacent to one another on said corresponding target nucleotide sequence, but are not ligated when the first and the second target-specific portions are hybridized with one or more mismatches to a nucleotide sequence present in said reaction mixture, wherein each of said one or more ligation products comprises a ligated sequence which includes (1) the first target-specific portion of the first oligonucleotide probe in a corresponding probe set and (2) the second target-specific portion of the second oligonucleotide probe in said corresponding probe set, or complements thereof; subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more amplification products, each amplification product comprising (1) the ligated sequence of a corresponding ligation product from which said amplification product is amplified and (2) an addressable array-specific portion which distinguishes said amplification product from other amplification products that comprise other ligated sequences or complements thereof; capturing said one or more amplification products to a solid support; and detecting the identities of the addressable array-specific portions in said captured amplification products to indicate the presence of one or more target nucleotide sequences in said reaction mixture.
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Specification