Concatameric ligation products: compositions, methods and kits for same
View Patent ↗The present teachings relate to methods, compositions, and kits for detecting one or more target polynucleotide sequences in a sample, and methods compositions and kits for forming concatameric ligation products. In some embodiments of the present teachings, oligonucleotides are hybridized to complementary target polynucleotides and are ligated together to form a concatameric ligation product. In some embodiments of the present teachings, the concatameric ligation product can be amplified, and the identity and quantity of the target polynucleotides determined based on sequence introduced in the ligation reaction. Some embodiments of the present teachings provide methods for removing unligated probes from the reaction mixture. Some embodiments of the present teachings provide for highly multiplexed detection, identification, and quantification of a plurality of target polynucleotides using a variety of analytical procedures.
1. A method of forming a concatameric ligation product comprising;
providing a target polynucleotide, a primary looped linker, a primary oligonucleotide, a secondary oligonucleotide, and a secondary looped linker;
performing, in any suitable order:
hybridizing the primary oligonucleotide and the secondary oligonucleotide to the target polynucleotide;
hybridizing the primary looped linker to the primary oligonucleotide;
hybridizing the secondary looped linker to the secondary oligonucleotide;
ligating the primary looped linker to the primary oligonucleotide;
ligating the primary oligonucleotide to the secondary oligonucleotide; and,
ligating the secondary oligonucleotide to the secondary looped linker; and,
forming a concatameric ligation product.
2. A method of forming a nuclease-resistant ligation product comprising;
providing a target polynucleotide, a primary looped linker, a primary oligonucleotide, a secondary oligonucleotide, and a secondary looped linker,
wherein the primary looped linker comprises a blocking moiety, the secondary looped linker comprise a blocking moiety, or the primary looped linker comprises a blocking moiety and the secondary looped linker comprises a blocking moiety;
performing, in any suitable order;
hybridizing the primary oligonucleotide and the secondary oligonucleotide to the target polynucleotide;
hybridizing the primary looped linker to the primary oligonucleotide;
hybridizing the secondary looped linker to the secondary oligonucleotide;
ligating the primary looped linker to the primary oligonucleotide;
ligating the primary oligonucleotide to the secondary oligonucleotide; and,
ligating the secondary oligonucleotide to the secondary looped linker; thereby
forming a nuclease-resistant ligation product; and,
optionally treating the nuclease-resistant ligation product with at least one nuclease wherein the nuclease resistant ligation product internal to the blocking moiety(s) is not degraded by the nuclease.
3. A method of determining a target polynucleotide comprising;
forming a concacatmeric ligation product according to the method of claim 1 , wherein the concatameric ligation product comprises a primary looped linker, a primary oligonucleotide, a secondary oligonucleotide, and a secondary looped linker;
measuring the concatameric ligation product; and,
determining the target polynucleotide.
4. A method of determining an allele at a single nucleotide polymorphism (SNP) locus comprising;
forming a concatameric ligation product according to the method of claim 1 , wherein the concatameric ligation product comprises a primary looped linker, a primary oligonucleotide, a secondary oligonucleotide, and a secondary looped linker, wherein the primary oligonucleotide comprises a 3′ discriminating nucleotide,
measuring the concatameric ligation product; and,
determining the identity of the allele at the SNP locus.
5. The method according to claim 4 wherein determining the concatameric ligation product comprises;
hybridizing a mobility probe to an amplified concatameric ligation product and eluting the hybridized mobility probe; and,
measuring the eluted mobility probe with a mobility dependent analysis technique.
6. The method according to claim 5 wherein the mobility dependent analysis technique is capillary electrophoresis.