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US 10,501,785 B2Granted
Reagents and methods for autoligation chain reaction
AssigneeRicardo Mancebo
InventorRicardo Mancebo
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Abstract
The invention relates to the amplification of specific target nucleic acids. The invention provides methods, reagents, and kits for carrying out such amplification via the autoligation chain reaction (ACR).
Claims40
A reagent composition for amplifying a target nucleic acid sequence comprising:
- a first forward oligonucleotide having a first bond-forming reactive moiety;
- a second forward oligonucleotide having a second bond-forming reactive moiety;
- a first reverse oligonucleotide having a third bond-forming reactive moiety;
- and
- a second reverse oligonucleotide having a fourth bond-forming reactive moiety;
- wherein the first forward oligonucleotide, the second forward oligonucleotide, the first reverse oligonucleotide, or the second reverse oligonucleotide is configured to specifically anneal with the target nucleic acid sequence;
- and wherein
- the first forward oligonucleotide comprises the first bond-forming reactive moiety that is an azide group conjugated to a 3′ terminus of the first forward oligonucleotide, and the second forward oligonucleotide comprises the second bond-forming reactive moiety that is an alkyne group conjugated to a 5′ terminus of the second forward oligonucleotide;
- or the first forward oligonucleotide comprises the first bond-forming reactive moiety that is an alkyne group conjugated to a 3′ terminus of the first forward oligonucleotide, and the second forward oligonucleotide comprises the second bond-forming reactive moiety that is an azide group conjugated to a 5′ terminus of the second forward oligonucleotide;
- the first reverse oligonucleotide comprises the third bond-forming reactive moiety that is an azide group conjugated to a 3′ terminus of the first reverse oligonucleotide, and the second reverse oligonucleotide comprises the fourth bond-forming reactive moiety that is an alkyne group conjugated to a 5′ terminus of the second reverse oligonucleotide;
- or the first reverse oligonucleotide comprises the third bond-forming reactive moiety that is an alkyne group conjugated to a 3′ terminus of the first reverse oligonucleotide, and the second reverse oligonucleotide comprises the fourth bond-forming reactive moiety that is an azide group conjugated to a 5′ terminus of the second reverse oligonucleotide;
- the first forward oligonucleotide and the second forward oligonucleotide are fully complementary to the target nucleic acid sequence and the first bond-forming reactive moiety and the second bond-forming reactive moiety are juxtaposed when annealed to the target nucleic acid sequence;
- and
- the first reverse oligonucleotide and the second reverse oligonucleotide are fully complementary to a complement of the target nucleic acid sequence and the third bond-forming reactive moiety and the fourth bond-forming reactive moiety are juxtaposed when annealed to the complement of the target nucleic acid sequence.
11 more elements
A kit for amplifying two or more target nucleic acid sequences, comprising:
- a first forward oligonucleotide having a first bond-forming reactive moiety;
- a second forward oligonucleotide having a second bond-forming reactive moiety;
- a first reverse oligonucleotide having a third bond-forming reactive moiety;
- and
- a second reverse oligonucleotide having a fourth bond-forming reactive moiety;
- wherein the first forward oligonucleotide, the second forward oligonucleotide, the first reverse oligonucleotide, or the second reverse oligonucleotide is configured to specifically anneal with a target nucleic acid sequence;
- and wherein
- the first forward oligonucleotide comprises the first bond-forming reactive moiety that is an azide group conjugated to a 3′ terminus of the first forward oligonucleotide, and the second forward oligonucleotide comprises the second bond-forming reactive moiety that is an alkyne group conjugated to a 5′ terminus of the second forward oligonucleotide;
- or the first forward oligonucleotide comprises the first bond-forming reactive moiety that is an alkyne group conjugated to a 3′ terminus of the first forward oligonucleotide, and the second forward oligonucleotide comprises the second bond-forming reactive moiety that is an azide group conjugated to a 5′ terminus of the second forward oligonucleotide;
- the first reverse oligonucleotide comprises the third bond-forming reactive moiety that is an azide group conjugated to a 3′ terminus of the first reverse oligonucleotide, and the second reverse oligonucleotide comprises the fourth bond-forming reactive moiety that is an alkyne group conjugated to a 5′ terminus of the second reverse oligonucleotide;
- or the first reverse oligonucleotide comprises the third bond-forming reactive moiety that is an alkyne group conjugated to a 3′ terminus of the first reverse oligonucleotide, and the second reverse oligonucleotide comprises the fourth bond-forming reactive moiety that is an azide group conjugated to a 5′ terminus of the second reverse oligonucleotide;
- the first forward oligonucleotide and the second forward oligonucleotide are fully complementary to the target nucleic acid sequence and the first bond-forming reactive moiety and the second bond-forming reactive moiety are juxtaposed when annealed to the target nucleic acid sequence;
- and
- the first reverse oligonucleotide and the second reverse oligonucleotide are fully complementary to a complement of the target nucleic acid sequence and the third bond-forming reactive moiety and the fourth bond-forming reactive moiety are juxtaposed when annealed to the complement of the target nucleic acid sequence.
11 more elements
Dependencies are inferred from the claim text.