DIRECT MULTIPLEX CHARACTERIZATION OF GENOMIC DNA
First Claim
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1. A method for detecting a target sequence comprising a first and second target domain in a sample, said method comprising:
- a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;
i) a first targeting domain;
ii) a second targeting domain;
iii) at least a first universal priming site; and
iv) a cleavage site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a ligase to form a closed circular probe;
c) cleaving said closed circular probe at said cleavage site to form a cleaved probe;
d) amplifying said cleaved probe to form a plurality of amplicons; and
e) detecting said amplicons to detect the presence of said target sequence in said sample.
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Abstract
The invention is directed to novel methods of multiplexing nucleic acid reactions, including amplification, detection and genotyping. The invention relies on the use of precircle probes that are circularized in the presence of the corresponding target nucleic acids, cleaved, and then amplified.
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Citations
47 Claims
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1. A method for detecting a target sequence comprising a first and second target domain in a sample, said method comprising:
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a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;
i) a first targeting domain;
ii) a second targeting domain;
iii) at least a first universal priming site; and
iv) a cleavage site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a ligase to form a closed circular probe;
c) cleaving said closed circular probe at said cleavage site to form a cleaved probe;
d) amplifying said cleaved probe to form a plurality of amplicons; and
e) detecting said amplicons to detect the presence of said target sequence in said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method for detecting a target sequence in a sample, said target sequence comprising a first and second target domain and a gap domain between said first and second target domains, said method comprising:
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a) hybridizing at least one of a plurality of precircle probes to said target sequence to form a plurality of first hybridization complexes, said precircle probes each comprising;
i) a first targeting domain;
ii) a second targeting domain;
iii) a detection domain;
iv) at least a first universal priming site;
v) a cleavage site; and
vi) a barcode sequence;
wherein said plurality of first and second targeting domains are complementary to said plurality of first and second target domains and said gap domain will hybridize to at least one of said plurality of detection domains;
b) contacting said plurality of first hybridization complexes with a ligase to form a plurality of closed circular probes;
c) cleaving said plurality of closed circular probes at said cleavage sites to form a plurality of cleaved probes;
d) amplifying said cleaved probes to form amplicons; and
e) detecting the presence of said amplicons to detect the presence of said plurality of target sequences in said sample. - View Dependent Claims (21, 22, 23, 24, 25)
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26. A method for detecting in a sample a plurality of target sequences, wherein each of said plurality of target sequences comprises first and second target domains, said method comprising:
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a) hybridizing said plurality of target sequences to a plurality of precircle probes to form a plurality of first hybridization complexes, each of said precircle probes comprising;
i) a first targeting domain;
ii) a second targeting domain;
iii) at least a first universal priming site;
iv) a cleavage site; and
v) a barcode;
wherein said plurality of first and second targeting domains hybridize to said plurality of first and second target domains;
b) contacting said plurality of first hybridization complexes with a ligase to form a plurality of closed circular probes;
c) cleaving said plurality of closed circular probes at said cleavage sites to form a plurality of cleaved probes;
d) amplifying said cleaved probes to form amplicons; and
e) detecting the presence of said amplicons to detect the presence of said plurality of target sequences in said sample. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33)
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34. A method for identifying the base at a detection position in a target sequence comprising a first and second target domain separated by a gap domain, said gap domain comprising said detection position, said method comprising:
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a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;
i) a 5′
first targeting domain;
ii) a 3′
second targeting domain;
iii) at least a first universal priming site; and
iv) a cleavage site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a polymerase and at least one interrogation dNTP to form an extended precircle probe;
c) contacting said first hybridization complex comprising said extended precircle probe and said target sequence with a ligase to form a closed circular probe;
d) cleaving said closed circular probe at said cleavage site to form a cleaved probe;
e) amplifying said cleaved probe to form a plurality of amplicons;
f) detecting the presence of said amplicons to detect the presence of said target sequence in said sample. - View Dependent Claims (35, 36)
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37. A method for amplifying a target sequence comprising a first and second target domain in a sample, said method comprising:
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a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;
i) a first targeting domain;
ii) a second targeting domain;
iii) at least a first universal priming site; and
iv) a cleavage site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a ligase to form a closed circular probe;
c) cleaving said closed circular probe at said cleavage site to form a cleaved probe; and
d) amplifying said cleaved probe. - View Dependent Claims (38, 39)
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40. A method for detecting a target sequence comprising a first and second target domain in a sample, said method comprising:
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a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;
i) a first targeting domain;
ii) a second targeting domain; and
iii) at least a first universal priming site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a ligase to form a closed circular probe;
c) contacting said closed circular probe at least a first universal primer, an extension enzyme and NTPs to form an extension product;
d) amplifying said extension product to form amplicons; and
e) detecting said amplicons to detect the presence of said target sequence in said sample. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47)
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Specification