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Publication Number
US-11180787-B2
Publication Date
2021-11-23
Expiration Date
Abstract
Methods for amplification of a target nucleic acid sequence comprising strand invasion are provided in which strand invasion occurs both at upstream and downstream regions of the target nucleic acid sequence. Further provided are kits and compositions suitable for use in such methods. The methods may comprise amplifying a target nucleic acid sequence comprising a region of unknown sequence, or determining the sequence of a target nucleic acid comprising a region of unknown sequence.
Core Innovation
An isothermal strand-invasion nucleic-acid amplification method amplifies a nucleic acid sequence of unknown sequence that is flanked by an upstream binding region and a downstream binding region. The upstream binding region comprises a first adaptor sequence and the downstream binding region comprises a second adaptor sequence, and the upstream and downstream binding regions are rendered single-stranded so that primer binding and primer-based amplification can occur.
The method contacts the nucleic acid comprising the unknown sequence with a first strand invasion oligonucleotide and a second strand invasion oligonucleotide. Each strand invasion oligonucleotide has non-extendible 3′ termini, and each binds to its corresponding adaptor sequence. Strand invasion occurs at two sites through binding of the first strand invasion oligonucleotide to the first adaptor sequence and the second strand invasion oligonucleotide to the second adaptor sequence, exposing primer-binding regions.
The method further uses primers comprising an upstream primer and a downstream primer, where the primers are capable of amplifying the nucleic acid sequence. The appropriate conditions are isothermal conditions and include the presence of a recombinase, and the upstream and downstream binding regions are present in opposing strands of the nucleic acid, with the strand invasion oligonucleotides configured to bind in an antiparallel orientation.
Claims Coverage
The independent claim coverage centers on one inventive core: an isothermal strand-invasion amplification method for an unknown sequence flanked by adaptor sequences, using non-extendible 3′-terminus strand invasion oligonucleotides and upstream/downstream primers under isothermal conditions with a recombinase.
Adaptor-flanked unknown nucleic-acid amplification in isothermal conditions
A method amplifying a nucleic acid sequence of unknown sequence flanked by an upstream binding region comprising a first adaptor sequence and a downstream binding region comprising a second adaptor sequence, wherein strand invasion renders the upstream and downstream binding regions single-stranded to allow binding of primers.
Dual strand invasion oligonucleotides with non-extendible 3′ termini
Contacting the nucleic acid with a first strand invasion oligonucleotide having a non-extendible 3′ terminus and a second strand invasion oligonucleotide having a non-extendible 3′ terminus, where the first strand invasion oligonucleotide binds to the first adaptor sequence and the second strand invasion oligonucleotide binds to the second adaptor sequence, enabling two-site strand invasion and exposure of primer-binding regions.
Upstream and downstream primers capable of amplifying the target
Using primers comprising an upstream primer and a downstream primer, where the primers are capable of amplifying the nucleic acid sequence, with strand invasion occurring at two sites through binding to the first adaptor sequence and the second adaptor sequence.
Opposing-strand, antiparallel invasion under isothermal conditions with recombinase
Using appropriate conditions that are isothermal conditions and include the presence of a recombinase, wherein the upstream and downstream binding regions are present in opposing strands of the nucleic acid and the first strand invasion oligonucleotide and the second strand invasion oligonucleotide are configured to bind in an antiparallel orientation.
The core inventive structure is dual-site adaptor-mediated strand invasion using non-extendible 3′ termini invasion oligonucleotides, combined with upstream/downstream primers for amplification under isothermal conditions with a recombinase, with an opposing-strand and antiparallel invasion configuration.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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