DETECTION OF NUCLEIC ACID SEQUENCE DIFFERENCES USING COUPLED LIGASE DETECTION AND POLYMERASE CHAIN REACTIONS
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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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Citations
24 Claims
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1-9. -9. (canceled)
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10. A method for identifying one or more different target nucleotide sequences comprising:
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providing a sample potentially containing one or more target nucleotide sequences comprising sequence differences; providing one or more oligonucleotide probe sets, each probe set comprising (a) a first oligonucleotide probe comprising a target-specific portion, a further portion, and a 5′
upstream primer-specific portion and (b) a second oligonucleotide probe comprising a target-specific portion and a 3′
downstream primer-specific portion, wherein the first and second oligonucleotide probes in a particular set are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when the first and second oligonucleotide probes are hybridized to any other nucleotide sequence present in the sample;providing a ligase; blending the sample, the one or more oligonucleotide probe sets, and the ligase to form a ligase reaction mixture; subjecting the ligase reaction mixture to one or more ligase reaction cycles to form a ligation product sequence comprising (a) the 5′
upstream primer specific portion, (b) the further portion, (c) the target-specific portions, and (d) the 3′
downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding oligonucleotide probe set is present in the sample;providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer; providing a polymerase; blending the ligase reaction mixture with the one or a plurality of oligonucleotide primer sets, and the polymerase to form a polymerase chain reaction mixture; subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles to form extension products comprising the ligation product sequence and/or complements thereof; and detecting the extension products to identify one or more target nucleotide sequences in the sample. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. 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, an upstream primer-specific portion, and a further portion; and
(b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence and a downstream primer-specific portion, 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 upstream primer-specific portion, (2) the further portion, (3) first and second target-specific portions and (4) the downstream-primer specific portion, or complements thereof;providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer; subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products comprising the ligation product sequences and/or complements thereof, and detecting the extension products to identify one or more target nucleotide sequences in the sample.
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24. 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, an upstream primer-specific portion, and a further portion; and
(b) a second oligonucleotide probe comprising a second target-specific portion capable of hybridizing to said corresponding target nucleotide sequence and a downstream primer-specific portion, 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 upstream primer-specific portion, (2) the further portion, (3) first and second target-specific portions and (4) the downstream-primer specific portion, or complements thereof;providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer;
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Specification