Template-dependent ligation with PNA-DNA chimeric probes
AssigneeApplera Corporation
InventorsMichael Egholm, Caifu Chen
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Abstract
The invention provides methods, kits, and compositions for ligation of PNA-DNA chimeric probes and oligonucleotides when they are hybridized adjacently to template nucleic acids using ligases and ligation reagents. Structural requirements of the chimeras for ligation include 5 to 15 contiguous PNA monomer units, 2 or more contiguous nucleotides, and a 3′ hydroxyl or 5′ hydroxyl terminus. The chimera and/or oligonucleotide may be labelled with fluorescent dyes or other labels. The methods include, for example, oligonucleotide-ligation assays (OLA) and single nucleotide polymorphism detection.
Claims39
A method of producing a template-dependent ligation product comprising the step of enzymatically ligating a PNA-DNA chimeric probe to a second probe in the presence of a template nucleic acid and a ligase, said chimeric probe having a PNA moiety and DNA moiety, said DNA moiety having at least two nucleotides and a 3′ hydroxyl or 5′ hydroxyl terminus,
- wherein the chimeric probe and the second probe are each hybridized to the template nucleic acid and adjacent to each other, and at least a portion of the PNA moiety is hybridized to the template, and wherein the second probe is a PNA-DNA chimera or an oligonucleotide.
The method of claim I in which the template nucleic acid is an RNA and the ligase is an RNA ligase.
A method of template-dependent ligation, comprising the steps of:
- a) generating a ligation product by enzymatically ligating a PNA-DNA chimeric probe to a second probe in the presence of a template to which the chimeric probe and the second probe are complementary and hybridized adjacently, wherein the second probe is a PNA-DNA chimera or an oligonucleotide;
- and
- b) detecting the ligation product.
A kit for template-dependent ligation comprising:
- a PNA-DNA chimeric probe, said probe comprising 5 to 15 contiguous PNA monomer units, 2 to 15 contiguous nucleotides, and a 3′ hydroxyl terminus;
- a second probe which is a PNA-DNA chimera or an oligonucleotide;
- and a ligase enzyme.
A duplex hybrid comprising:
- a PNA-DNA chimeric probe, a second probe which is a PNA-DNA chimera or an oligonucleotide, and a template nucleic acid with a sequence complementary to the chimeric probe or containing one or more mismatches relative to the chimeric probe,
- wherein a terminus of the second probe hybridizes adjacent to a terminus of the chimeric probe on the template and said terminii can be ligated together by a ligase enzyme.
Dependencies are inferred from the claim text.