Amplification of nucleic acids with electronic detection
First Claim
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1. A method for detecting a target sequence in a sample comprising:
- a) providing a rolling circle probe (RCP) comprising;
i) a first ligation sequence substantially complementary to a first domain of said target sequence;
ii) a second ligation sequence substantially complementary to a second domain of said target sequence; and
iii) a priming sequence;
b) hybridizing said first ligation sequence to said first domain and said second ligation sequence to said second domain to form a first hybridization complex;
c) ligating said first and second ligation sequences together;
d) adding to said first hybridization complex;
i) a primer substantially complementary to said priming sequence;
ii) a polymerase;
iii) dNTPs; and
iv) an electron transfer moiety (ETM);
to form a rolling circle concatamer comprising at least one covalently attached ETM; and
e) detecting said ETM as an indicator of the presence of said target sequence.
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Abstract
The invention relates to compositions and methods useful in the detection of nucleic acids using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the nucleic acid, either directly or indirectly, to allow electronic detection of the ETM using an electrode.
190 Citations
23 Claims
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1. A method for detecting a target sequence in a sample comprising:
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a) providing a rolling circle probe (RCP) comprising;
i) a first ligation sequence substantially complementary to a first domain of said target sequence;
ii) a second ligation sequence substantially complementary to a second domain of said target sequence; and
iii) a priming sequence;
b) hybridizing said first ligation sequence to said first domain and said second ligation sequence to said second domain to form a first hybridization complex;
c) ligating said first and second ligation sequences together;
d) adding to said first hybridization complex;
i) a primer substantially complementary to said priming sequence;
ii) a polymerase;
iii) dNTPs; and
iv) an electron transfer moiety (ETM);
to form a rolling circle concatamer comprising at least one covalently attached ETM; and
e) detecting said ETM as an indicator of the presence of said target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
a) cleaving said concatamer to form RCP amplicons, each of which comprises a covalently attached ETM and a capture sequence; and
b) hybridizing said capture sequence to a capture probe covalently attached to an electrode.
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4. A method according to claim 1 wherein said ETM is covalently attached to at least one of said dNTPs.
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5. A method according to claim 2 wherein said electrode further comprises a self-assembled monolayer.
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6. A method according to claim 5 wherein said self-assembled monolayer comprises insulators.
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7. A method according to claim 1 wherein said first and second target domains are directly adjacent.
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8. A method according to claim 1 wherein said first and second target domains are separated by one or more nucleotides.
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9. A method according to claim 1 wherein said RCP comprises at least one nucleotide analog.
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10. A method according to claim 1 wherein said primer hybridizes both to said target sequence and to said priming sequence.
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11. A method according to claim 1 wherein said cleavage site comprises uracil.
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12. A method for detecting a first target nucleic acid sequence comprising:
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a) hybridizing at least a first primer nucleic acid to said first target sequence to form a first hybridization complex;
b) contacting said first hybridization complex with a first enzyme to form a modified first primer nucleic acid;
c) disassociating said first hybridization complex;
d) forming a first assay complex comprising at least one electron transfer moiety (ETM) and said modified first primer nucleic acid, wherein said first assay complex is covalently attached to an electrode;
wherein said electrode is on a supporting substrate, and said substrate comprises a plurality of electrodes; and
e) detecting electron transfer between said ETM and said electrode as an indication of the presence of said target sequence. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
a) hybridizing at least a second primer nucleic acid to a second target sequence that is substantially complementary to said first target sequence to form a second hybridization complex;
b) contacting said second hybridization complex with said first enzyme to form a modified second primer nucleic acid;
c) disassociating said second hybridization complex; and
d) forming a second assay complex comprising at least one ETM and said modified second primer nucleic acid wherein said second assay complex is covalently attached to a second electrode.
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15. A method according to claim 12 wherein steps a) through c) are repeated prior to step d).
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16. A method according to claim 13 wherein said first enzyme comprises a DNA polymerase and said modification is an extension of said primer such that the polymerase chain reaction (PCR) occurs.
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17. A method according to claim 13 wherein said first enzyme comprises a ligase and said modification comprises a ligation of said first primer which hybridizes to a first domain of said first target sequence to a second primer which hybridizes to a second adjacent domain of said first target sequence, such that the ligase chain reaction (LCR) occurs.
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18. A method according to claim 14 wherein said first enzyme comprises a ligase and said modification is a ligation of said second primer which hybridizes to a first domain of said second target sequence to a fourth primer which hybridizes to a second adjacent domain of said second target sequence, such that the ligase chain reaction (LCR) occurs.
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19. A method according to claim 13 wherein said first primer comprises a first probe sequence, a first scissile linkage and a second probe sequence, wherein said first enzyme will cleave said first scissile linkage resulting in the separation of said first and said second probe sequences and the disassociation of said first hybridization complex, leaving said first target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
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20. A method according to claim 15 wherein said second primer comprises a third probe sequence, a second scissile linkage and a fourth probe sequence, wherein said first enzyme will cleave said second scissile linkage resulting in the separation of said third and said fourth probe sequences and the disassociation of said second hybridization complex, leaving said second target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
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21. A method according to claim 13 wherein said first enzyme is a polymerase that extends said first primer and said modified first primer comprises a first newly synthesized strand, and said method further comprises:
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a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended first primer leaving said first target sequence intact; and
b) extending from said nick using said polymerase, thereby displacing said first newly synthesized strand and generating a second newly synthesized strand;
such that strand displacement amplification (SDA) occurs.
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22. A method according to claim 15 wherein said first enzyme is a polymerase that extends a second primer and said modified first primer comprises a third newly synthesized strand, and said method further comprises:
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a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended second primer leaving said second target sequence intact; and
b) extending from said nick using said polymerase, thereby displacing said third newly synthesized strand and generating a fourth newly synthesized strand;
such that strand displacement amplification (SDA) occurs.
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23. A method according to claim 12 wherein said first primer is an invader primer, said method further comprises hybridizing a signaling primer to said target sequence, said enzyme comprises a structure-specific cleaving enzyme and said modification comprises a cleavage of said signaling primer, such that the Invader™
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Specification