Nucleic acid amplification method
First Claim
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1. A method of generating circularized nucleic acid, said method comprising:
- providing a single-stranded target nucleic acid;
hybridizing the single-stranded target nucleic acid with a circularization adaptor having(i) single-stranded sequences at each end and on the same strand, which single-stranded sequences are complementary to the ends of the single-stranded target nucleic acid and(ii) a double-stranded non-target complementary segment between said single-stranded end sequences; and
circularizing the target nucleic acid using a DNA ligase.
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Abstract
A nucleic acid amplification method, and probes for use within the method are described.
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Citations
22 Claims
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1. A method of generating circularized nucleic acid, said method comprising:
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providing a single-stranded target nucleic acid; hybridizing the single-stranded target nucleic acid with a circularization adaptor having (i) single-stranded sequences at each end and on the same strand, which single-stranded sequences are complementary to the ends of the single-stranded target nucleic acid and (ii) a double-stranded non-target complementary segment between said single-stranded end sequences; and circularizing the target nucleic acid using a DNA ligase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. 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 circularization adaptor having single-stranded sequences at each end complementary to the ends of the target fragment and, between said single-stranded end sequences, a double-stranded non-target-complementary segment, and (iii) amplifying said circularized target nucleic acid fragment.
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10. A method of analyzing at least one circularized nucleic acid, the method comprising amplifying the said circularized nucleic acid to provide an amplification product free in solution which comprises a concatemer of a sequence to be analysed coiled into a ball, the method further comprising the step of:
directly detecting by microscopic analysis said concatemer amplification product ball which is free in solution in a homogeneous detection reaction by hybridizing hybridization probes being fluorescence-labelled oligonucleotides to said amplification product ball, wherein the hybridization of the oligonucleotides results in an enrichment of the oligonucleotides in said amplification product ball and said enrichment causes a condensed signal which can be directly detected by microscopic analysis due to the contrast between said condensed signal and the diffuse signal from non-bound oligonucleotides. - View Dependent Claims (11, 12, 13, 14, 15)
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16. A method for generating a circularized nucleic acid molecule from two oligonucleotides, being parts of a pair of proximity probes, that have been joined in a proximity-dependent nucleic acid ligation reaction to form a joined product, said method comprising:
hybridizing a restriction oligonucleotide to two identical sequences present in both of the joined oligonucleotides in said joined product, digesting said joined product at the restriction sites thus created to release a linear nucleic acid molecule, and circularizing 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 (17, 18, 19, 20, 21, 22)
Specification