Coupled amplification and ligation method
AssigneePerkin Elmer - Applied Biosystems Division
InventorFaye Eggerding
Life of this patent
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Abstract
A method based on polymerase chain reaction (PCR) amplification and oligonucleotide ligase assay (OLA) reaction is provided for analyzing complex genetic systems in a single reaction vessel. The method involves simultaneously incubating a sample containing one or more target polynucleotides with PCR primers and OLA probes in a single reaction mixture. The presence of variant polynucleotide sequences in the sample is determined by detecting and identifying the products of the OLA reaction.
Claims28
A method for detecting a target polynucleotide in a sample comprising:
- amplifying the target polynucleotide by primer extension to form an amplification product that is complementary to the target polynucleotide, the amplification being performed (a) in the presence of at least one pair of oligonucleotide probes capable of hybridizing to contiguous sequences of the amplification product, and (b) at a higher temperature than a melting point temperature of the oligonucleotide probes;
- performing multiple ligation cycles during which oligonucleotide probes which hybridize to contiguous sequences of the amplification product are ligated to form an oligonucleotide probe ligation product;
- and
- detecting the oligonucleotide probe ligation product.
A method for detecting a plurality of target polynucleotides in a sample comprising:
- amplifying the plurality of target polynucleotides by primer extension to form a plurality of amplification products that are complementary to the plurality of target polynucleotides, the amplification being performed (a) in the presence of a plurality of pairs of oligonucleotide probes capable of hybridizing to contiguous sequences of the amplification products, and (b) at a higher temperature than a melting point temperature of the oligonucleotide probes;
- performing multiple ligation cycles during which oligonucleotide probes which hybridize to contiguous sequences of the amplification products are ligated to form oligonucleotide probe ligation products;
- and
- detecting the plurality of oligonucleotide probe ligation products.
A method for detecting a plurality of target polynucleotides in a sample comprising:
- performing a first ligation using a first set of oligonucleotide probes that are complementary to the plurality of target polynucleotides to form first ligation products that are complementary to the plurality of target polynucleotides, the first ligation being performed (a) in the presence of a plurality of pairs of a second set of oligonucleotide probes capable of hybridizing to contiguous sequences of the first ligation products, and (b) at a higher temperature than a melting point temperature of the second set of oligonucleotide probes;
- performing a second ligation using those members of the second set of oligonucleotide probes which hybridize to contiguous sequences of the first ligation products to form second ligation products;
- and
- detecting the second ligation products.
A kit for detecting a target polynucleotide of known sequence in a sample comprising:
- a sufficient quantity of ligation probes to perform a ligation-based amplification reaction on the target polynucleotide to form a ligation-based amplification product;
- and
- a sufficient quantity of two isolated oligonucleotide probes capable of hybridizing to contiguous sequences of the amplification product to perform a ligation reaction on the oligonucleotide probes, the ligation probes having a melting point temperature sufficiently high to enable performance of the ligation-based amplification reaction at a temperature above the melting point temperature of the oligonucleotide probes.
Dependencies are inferred from the claim text.