Detection of nucleic acid sequence differences using coupled ligase detection with padlock probes and polymerase chain reactions
First Claim
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1. A method for identifying one or more different target nucleotide sequences comprising:
- providing a sample potentially containing one or more target nucleotide sequences comprising sequence differences;
providing one or more padlock oligonucleotide probes, each probe comprising (a) a first target-specific portion and a 5′
upstream primer-specific portion and (b) a second target-specific portion and a 3′
downstream primer-specific portion, wherein the first and second target-specific portions of a particular probe are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when first and second target-specific portions of a particular probe are hybridized to any other nucleotide sequence present in the sample;
providing a ligase;
blending the sample, the one or more padlock oligonucleotide probes, and the ligase to form a ligase detection reaction mixture;
subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles to form a ligation product sequence comprising (a) the 5′
upstream primer specific portion, (b) the target-specific portions, and (c) the 3′
downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding padlock oligonucleotide probe 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 detection 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 identity one or more target nucleotide sequences in the sample.
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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
17 Claims
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1. 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 padlock oligonucleotide probes, each probe comprising (a) a first target-specific portion and a 5′
upstream primer-specific portion and (b) a second target-specific portion and a 3′
downstream primer-specific portion, wherein the first and second target-specific portions of a particular probe are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when first and second target-specific portions of a particular probe are hybridized to any other nucleotide sequence present in the sample;providing a ligase; blending the sample, the one or more padlock oligonucleotide probes, and the ligase to form a ligase detection reaction mixture; subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles to form a ligation product sequence comprising (a) the 5′
upstream primer specific portion, (b) the target-specific portions, and (c) the 3′
downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding padlock oligonucleotide probe 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 detection 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 identity one or more target nucleotide sequences in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for identifying one or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or trans locations 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 padlock oligonucleotide probes, each said probe including (a) a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and an upstream primer-specific portion and (b) 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 first target-specific portion of the padlock oligonucleotide probe and (2) the second target-specific portion of the padlock oligonucleotide probe, 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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17. 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 padlock oligonucleotide probes, each said probe including (a) a first target-specific portion capable of hybridizing to a corresponding target nucleotide sequence and an upstream primer-specific portion and (b) 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 target-specific portions of the padlock oligonucleotide probes and (3) 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, wherein one of the primers has an addressable array-specific portion; subjecting said one or more ligation products to one or more polymerase chain reaction cycles to produce one or more extension products, each extension product comprising (1) the ligated sequence of a corresponding ligation product from which said extension product is amplified and (2) an addressable array-specific portion which distinguishes said extension product from other extension products that comprise other ligated sequences or complements thereof; capturing said one or more extension products to a solid support; and detecting the identities of the addressable array-specific portions in said captured extension products to indicate the presence of one or more target nucleotide sequences in said reaction mixture.
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Specification