Compositions and methods for rolling circle amplification
First Claim
Patent Images
1. A method of detecting the presence of a target sequence comprising:
- a) providing a device comprising a substrate having a capture probe comprising;
i) a first domain substantially complementary to a open circle probe; and
ii) a second domain comprising a cleavage site;
wherein said capture probe is attached to said substrate at both termini;
b) contacting said capture probe with said target sequence and said open circle probe to form a hybridization complex;
c) contacting said hybridization complex with a ligase such that said open circle probe circularizes to form a second hybridization complex;
d) contacting said capture probe with a cleavage agent to cleave said probe at said cleavage site;
e) adding an extension enzyme and NTPs to said second hybridization complex to form an extended capture probe; and
f) detecting said extended capture probe.
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Abstract
This invention is directed to novel methods of amplifying and detecting DNA. More specifically, the invention applies variations of Rolling Circle Amplification to several detection platforms.
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Citations
33 Claims
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1. A method of detecting the presence of a target sequence comprising:
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a) providing a device comprising a substrate having a capture probe comprising;
i) a first domain substantially complementary to a open circle probe; and
ii) a second domain comprising a cleavage site;
wherein said capture probe is attached to said substrate at both termini;
b) contacting said capture probe with said target sequence and said open circle probe to form a hybridization complex;
c) contacting said hybridization complex with a ligase such that said open circle probe circularizes to form a second hybridization complex;
d) contacting said capture probe with a cleavage agent to cleave said probe at said cleavage site;
e) adding an extension enzyme and NTPs to said second hybridization complex to form an extended capture probe; and
f) detecting said extended capture probe. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of detecting the presence of a target sequence comprising a first and a second target domain, said method comprising:
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a) providing a device comprising a substrate comprising a capture probe substantially complementary to a first domain of said target sequence;
b) contacting said capture probe with;
i) said target sequence; and
ii) a rolling circle primer comprising;
1) a first domain substantially complementary to said second domain of said target sequence; and
2) a second domain substantially complementary to a first domain of a circularized probe;
to form a hybridization complex;
c) contacting said hybridization complex with a ligase such that capture probe and said rolling circle primer ligate;
d) hybridizing said second domain of said rolling circle primer to a circularized probe to form a second hybridization complex;
e) adding an extension enzyme and NTPs to said second hybridization complex to form an extended capture probe; and
f) detecting said extended capture probe. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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17. A method of detecting the presence of a target sequence comprising a first target domain adjacent to a second target domain, said method comprising:
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a) providing a device comprising a substrate comprising a capture probe substantially complementary to said second target domain of said target sequence;
b) contacting said capture probe with;
i) said target sequence; and
ii) a ligation probe comprising;
1) a first domain substantially complementary to said second domain of said target sequence; and
2) a rolling circle primer;
wherein when the nucleotides at the adjacent termini of said capture probe and said ligation probe are perfectly complementary to the respective target nucleotides, said capture probe and said ligation probe are ligated to form a ligated probe;
c) hybridizing said rolling circle primer of said ligated probe with a rolling circle priming sequence of a closed circle probe to form a rolling circle hybridization structure;
d) providing an extension enzyme and NTPs such that said ligated probe is extended; and
e) detecting said extended ligated probe. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24)
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25. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising:
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a) providing a first hybridization complex comprising said target sequence and a capture probe comprising an interrogation position;
b) contacting said first hybridization complex with an extension enzyme and at least one chain terminating nucleotriphosphate comprising a hapten, under conditions wherein only if the nucleotides at said detection and interrogation positions are perfectly complementary does said capture probe get extended;
c) adding;
i) secondary probe comprising;
1) the binding partner of said hapten; and
2) a rolling circle primer;
ii) closed circle probe comprising a rolling circle priming sequence;
to form a second hybridization complex between said rolling circle primer and said rolling circle priming sequence;
d) contacting said second hybridization complex with an extension enzyme and NTPs to extend said primer; and
e) detecting said extended primer. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32, 33)
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Specification