NUCLEIC ACID AMPLIFICATION METHOD
First Claim
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1. A method of generating a circularised nucleic acid, said method comprising fragmenting a target nucleic acid and then circularising a target nucleic acid fragment thus obtained using a circularisation adaptor having single-stranded sequences at each end complementary to the ends of the target fragment.
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Abstract
A nucleic acid amplification method, and probes for use within the method are described.
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Citations
21 Claims
- 1. A method of generating a circularised nucleic acid, said method comprising fragmenting a target nucleic acid and then circularising a target nucleic acid fragment thus obtained using a circularisation adaptor having single-stranded sequences at each end complementary to the ends of the target fragment.
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10. A method of amplifying a target nucleic acid fragment, said method comprising:
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(i) fragmenting a target nucleic acid, (ii) circularizing a target nucleic acid fragment thus obtained, using a circularisation adaptor having single-stranded sequences at each end complementary to the ends of the target fragment, and (iii) amplifying said circularised target nucleic acid fragment.
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11. A method of analyzing a circularised nucleic acid, the method comprising amplifying the said circularised nucleic acid to provide an amplification product which comprises a concatemer of a sequence to be analysed, the method further comprising the step of:
directly detecting the said concatemer amplification product in a homogeneous detection reaction by hybridising signaling probes to said amplification product, wherein the hybridisation of the probes results in an enrichment of the probes in the said amplification product, allowing the product to be detected directly in a homogeneous reaction. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18)
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19. A method for generating a circularised nucleic acid molecule from oligonucleotide probes that have been ligated in a proximity-dependent nucleic acid ligation reaction to form a joined probe molecule, said method comprising:
hybridising a restriction oligonucleotide to two identical sequences in two of the joined oligonucleotide probes in said joined probe molecule, digesting said joined molecule at the restriction sites thus created to release a linear nucleic acid molecule, and circularising said released linear nucleic acid molecule by ligation by using an intact restriction oligonucleotide as a ligation template, said intact restriction oligonucleotide being complementary to both ends of said released linear molecule. - View Dependent Claims (20, 21)
Specification