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US 6,686,150 B1Granted
Amplification of nucleic acids with electronic detection
AssigneeClinical Micro Sensors, Inc.
InventorsGary Blackburn, Bruce D. Irvine, Jon Faiz Kayyem, Edward Lewis Sheldon, III, Robert H. Terbrueggen
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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.
Claims23
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.
13 more elements
A method for detecting a first target nucleic acid sequence comprising:
- 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.
4 more elements
Dependencies are inferred from the claim text.