Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
DC CAFCFirst Claim
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 oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe comprising a target-specific 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 each 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 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 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 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 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 identify one or more target nucleotide sequences in the sample.
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Abstract
The present invention relates to the detection of nucleic acid sequence differences using coupled ligase detection reaction and polymerase chain reaction. One aspect of the present invention involves use of a ligase detection reaction coupled to a polymerase chain reaction. Another aspect of the present invention relates to the use of a primary polymerase chain reaction coupled to a secondary polymerase chain reaction coupled to a ligase detection reaction. A third aspect of the present invention involves a primary polymerase chain reaction coupled to a secondary polymerase chain reaction. 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
22 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 oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe comprising a target-specific 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 each 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 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 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 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 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 identify 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, 16, 17, 18, 19, 20, 21, 22)
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Specification