Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
First Claim
1. 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:
- providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;
providing one or more oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and a 5′
upstream primer-specific portion and (b) a second oligonucleotide probe, having a target-specific portion and a 3′
downstream primer-specific portion, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample;
providing a ligase;
blending the sample, the plurality of 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 comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligation product sequence containing (a) the 5′
upstream primer-specific portion, (b) the target-specific portions connected together, and (c) the 3′
downstream primer-specific portion with the ligation product sequence for each set being distinguishable from other nucleic acids in the ligase detection reaction mixture, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment;
providing one or a plurality of oligonucleotide primer sets, each set characterized by (a) an upstream primer containing the same sequence as the 5′
upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3′
downstream primer-specific portion of the ligation product sequence, wherein one of the primers has a detectable reporter label;
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 comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the primers hybridize to their complementary primer-specific portions of the ligation product sequence, and an extension treatment, wherein the hybridized primers are extended to form extension products complementary to the sequences to which the primers are hybridized, wherein, in a first cycle, the downstream primer hybridizes to the 3′
downstream primer-specific portion of the ligation product sequence and extends to form an extension product complementary to the ligation product sequence, and, in subsequent cycles, the upstream primer hybridizes to the 5′
upstream primer-specific portion of the extension product complementary to the ligation product sequence and the 3′
downstream primer hybridizes to the 3′
downstream portion of the ligation product sequence;
detecting the reporter labels; and
distinguishing the extension products to indicate the presence of 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.
23 Citations
54 Claims
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1. 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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providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;
providing one or more oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a target-specific portion and a 5′
upstream primer-specific portion and (b) a second oligonucleotide probe, having a target-specific portion and a 3′
downstream primer-specific portion, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample;
providing a ligase;
blending the sample, the plurality of 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 comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the target nucleotide sequences, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligation product sequence containing (a) the 5′
upstream primer-specific portion, (b) the target-specific portions connected together, and (c) the 3′
downstream primer-specific portion with the ligation product sequence for each set being distinguishable from other nucleic acids in the ligase detection reaction mixture, and, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences in the sample other than their respective target nucleotide sequences but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment;
providing one or a plurality of oligonucleotide primer sets, each set characterized by (a) an upstream primer containing the same sequence as the 5′
upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3′
downstream primer-specific portion of the ligation product sequence, wherein one of the primers has a detectable reporter label;
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 comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the primers hybridize to their complementary primer-specific portions of the ligation product sequence, and an extension treatment, wherein the hybridized primers are extended to form extension products complementary to the sequences to which the primers are hybridized, wherein, in a first cycle, the downstream primer hybridizes to the 3′
downstream primer-specific portion of the ligation product sequence and extends to form an extension product complementary to the ligation product sequence, and, in subsequent cycles, the upstream primer hybridizes to the 5′
upstream primer-specific portion of the extension product complementary to the ligation product sequence and the 3′
downstream primer hybridizes to the 3′
downstream portion of the ligation product sequence;
detecting the reporter labels; and
distinguishing the extension products to indicate the presence of 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, 23, 24, 25, 26, 27, 28)
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29. A method for identifying two 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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providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;
providing one or more of primary oligonucleotide primer groups, each group comprised of one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′
upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′
upstream secondary primer-specific portion, wherein the first oligonucleotide primers of each set in the same group contain the same 5′
upstream secondary primer-specific portion and the second oligonucleotide primers of each set in the same group contain the same 5′
upstream secondary primer-specific portion, wherein the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product, but have a mismatch which interferes with formation of such a polymerase chain reaction product when hybridized to any other nucleotide sequence present in the sample, and wherein the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products in the same group or other groups;
providing a polymerase;
blending the sample, the primary oligonucleotide primers, and the polymerase to form a primary polymerase chain reaction mixture;
subjecting the primary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the target-specific portions of the primary oligonucleotide primers hybridize to the target nucleotide sequences, and an extension treatment, wherein the hybridized primary oligonucleotide primers are extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primer is hybridized;
providing one or a plurality of secondary oligonucleotide primer sets, each set characterized by (a) a first secondary primer containing the same sequence as the 5′
upstream portion of a first primary oligonucleotide primer, and (b) a second secondary primer containing the same sequence as the 5′
upstream portion of a second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide contained by the first secondary primer, wherein a set of secondary oligonucleotide primers may be used to amplify all of the primary extension products in a given group;
blending the primary extension products, the secondary oligonucleotide primers, and the polymerase to form a secondary polymerase chain reaction mixture;
subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the secondary oligonucleotide primers hybridize to the primary extension products, an extension treatment, wherein the hybridized secondary oligonucleotide primers are extended to form secondary extension products complementary to the primary extension products;
providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a secondary extension product-specific portion and a detectable reporter label, and (b) a second oligonucleotide probe, having a secondary extension product-specific portion, wherein the oligonucleotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a complementary secondary extension product-specific portion, but have a mismatch which interferes with such ligation when hybridized to any other nucleotide sequence present in the sample;
providing a ligase;
blending the secondary extension products, the plurality of 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 comprising a denaturation treatment, wherein any hybridized oligonucleotides are separated from the secondary extension product, and a hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective secondary extension products, if present, and ligate to one another to form a ligation product sequence containing (a) the detectable reporter label and (b) the secondary extension product-specific portions connected together, wherein the oligonucleotide probe sets may hybridize to nucleotide sequences other than their respective complementary secondary extension products but do not ligate together due to a presence of one or more mismatches and individually separate during the denaturation treatment, and detecting the reporter labels of the ligation product sequences, thereby indicating the presence of two or more target nucleotide sequences in the sample. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
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51. A method for identifying two 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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providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;
providing one or more primary oligonucleotide primer groups, each group comprised of one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′
upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′
upstream secondary primer-specific portion, wherein the first oligonucleotide primers of each set in the same group contain the same 5′
upstream secondary primer-specific portion and the second oligonucleotide primers of each set in the same group contain the same 5′
upstream secondary primer-specific portion, wherein the oligonucleotide primers in a particular set are suitable for hybridization on complementary strands of a corresponding target nucleotide sequence to permit formation of a polymerase chain reaction product, but have a mismatch which interferes with formation of such a polymerase chain reaction product when hybridized to any other nucleotide sequence present in the sample, and wherein the polymerase chain reaction products in a particular set may be distinguished from other polymerase chain reaction products in the same group or other groups;
providing a polymerase;
blending the sample, the primary oligonucleotide primers, and the polymerase to form a primary polymerase chain reaction mixture;
subjecting the primary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic acid sequences are separated, a hybridization treatment, wherein the target-specific portion of the primary oligonucleotide primers hybridize to the target nucleotide sequences, and an extension treatment, wherein the hybridized primary oligonucleotide primers are extended to form primary extension products complementary to the target nucleotide sequence to which the primary oligonucleotide primer is hybridized;
providing one or a plurality of secondary oligonucleotide primer sets, each set characterized by (a) a first secondary primer, having a detectable reporter label and containing the same sequence as the 5′
upstream portion of a first primary oligonucleotide primer, and (b) a second secondary primer containing the same sequence as the 5′
upstream portion of a second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide complementary to the first secondary primer, wherein a set of secondary oligonucleotide primers amplify the primary extension products in a given group;
blending the primary extension products, the secondary oligonucleotide primers, and the polymerase to form a secondary polymerase chain reaction mixture;
subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles comprising a denaturation treatment, wherein hybridized nucleic sequences are separated, a hybridization treatment, wherein the secondary oligonucleotide primers hybridize to the primary extension products, an extension treatment, wherein the hybridized secondary oligonucleotide primers are extended to form secondary extension products complementary to the primary extension product; and
detecting the labeled secondary extension products, thereby indicating the presence of one or more target nucleotide sequences in the sample. - View Dependent Claims (52, 53, 54)
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Specification