Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
First Claim
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1. A method comprising:
- providing a sample potentially containing one or more target nucleotide sequences;
forming primary products in a reaction process comprising a polymerase reaction, wherein each primary product comprises a 5′
primer-specific portion, a target nucleotide sequence-specific portion, and a 3′
primer-specific portion, wherein the 5′
primer-specific portion and the 3′
primer-specific portion differ from each other and are not the same as or complementary to the target-specific portion;
providing one or more oligonucleotide primer sets, each oligonucleotide primer set characterized by a first and second oligonucleotide primer, wherein said first oligonucleotide primer is capable of hybridizing to the 3′
primer-specific portion of a primary product and said second oligonucleotide primer is capable of hybridizing to a 3′
primer specific portion of a complement of the primary product; and
forming secondary products in a polymerase reaction mixture comprising the one or more oligonucleotide primer sets, wherein said secondary products are complementary to each of the formed primary products or complements thereof.
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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 ligation 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 difference.
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Citations
16 Claims
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1. A method comprising:
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providing a sample potentially containing one or more target nucleotide sequences; forming primary products in a reaction process comprising a polymerase reaction, wherein each primary product comprises a 5′
primer-specific portion, a target nucleotide sequence-specific portion, and a 3′
primer-specific portion, wherein the 5′
primer-specific portion and the 3′
primer-specific portion differ from each other and are not the same as or complementary to the target-specific portion;providing one or more oligonucleotide primer sets, each oligonucleotide primer set characterized by a first and second oligonucleotide primer, wherein said first oligonucleotide primer is capable of hybridizing to the 3′
primer-specific portion of a primary product and said second oligonucleotide primer is capable of hybridizing to a 3′
primer specific portion of a complement of the primary product; andforming secondary products in a polymerase reaction mixture comprising the one or more oligonucleotide primer sets, wherein said secondary products are complementary to each of the formed primary products or complements thereof. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method comprising:
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providing a sample potentially containing one or more target nucleotide sequences and complements thereof; adding a 5′
primer-specific portion and a 3′
primer-specific portion, which differ from each other, to the one or more target nucleotide sequences and complements thereof in the sample in a reaction process comprising a polymerase reaction to form one or more primer labeled target nucleotide sequences and complements thereof; andforming extension products that are complementary to the one or more primer labeled target nucleotide sequences and complements thereof in a polymerase reaction mixture comprising one or more oligonucleotide primer sets, each oligonucleotide primer set characterized by a first and second oligonucleotide primer, wherein said first oligonucleotide primer is capable of hybridizing to a 3′
primer-specific portion of a primer labeled target nucleotide sequence and said second oligonucleotide primer is capable of hybridizing to a 3′
primer specific portion of the complement of the primer labeled target nucleotide sequence. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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Specification