Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
First Claim
1. 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:
- 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;
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 to form primary extension products complementary to the target nucleotide sequence;
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 a polymerase to form a secondary polymerase chain reaction mixture;
subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles 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;
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 to form a ligation product sequence comprising (a) the detectable reporter label and (b) the secondary extension product-specific portions; and
detecting the reporter labels of the ligation product sequences, thereby indicating the presence of two 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
25 Claims
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1. 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;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 to form primary extension products complementary to the target nucleotide sequence; 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 a polymerase to form a secondary polymerase chain reaction mixture; subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles 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; 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 to form a ligation product sequence comprising (a) the detectable reporter label and (b) the secondary extension product-specific portions; 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 (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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23. A method comprising:
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providing a sample potentially containing one or more target nucleotide sequences comprising 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;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 to form primary extension products; 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 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 a polymerase to form a secondary polymerase chain reaction mixture; and subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles to form secondary extension products. - View Dependent Claims (24, 25)
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Specification