Amplification of nucleic acids with electronic detection
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.
1 . A method for detecting the presence of a ribosomal RNA target sequence in a nucleic acid sample, comprising:
a) applying an electronic input signal to a surface comprising an array of electrodes wherein said electrodes comprise a hybridization complex and said hybridization complex comprises:
i) a capture probe;
ii) an electron transfer moiety; and
ii) a ribosomal RNA target nucleic acid;
b) detecting the presence of said ribosomal RNA target sequence via an output signal.
2 . The method of claim 1 wherein said electron transfer moiety of said hybridizaton complex is attached to a label probe.
3 . The method of claim 1 wherein said electrodes further comprise a self assembled monolayer.
4 . The method of claim 1 wherein said electronic input signal is an AC input signal.
5 . The method of claim 1 wherein said electronic input signal is an AC/DC input signal.
6 . The method of claim 1 wherein said ribosomal RNA target nucleic acid is contacted with at least one helper probe prior to formation of said hybridization complex.
7 . The method of claim 1 wherein said ribosomal RNA target nucleic acid is selected from the group consisting of: 5S rRNA; 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA.
8 . The method of claim 1 wherein said rRNA target nucleic acid is from a bacteria selected from the group consisting of: Mycobacterium avium, Mycobacterium intracellulare, Legionella, Salmonella, Campylobactor, Proteus mirabilis, Enterococcus, Enterobacter cloacae, Escherichia coli, Nisseria gonorrhoeae, Nisseria meningitidis, Chlamydia trachomatis, Chlamydia pssittici, Chlamydia pneumoniae, Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus hominus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma Orale, Mycoplasma faucium, Mycoplasma buccale, Mycoplasma salivarium, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus bovis, Haemophilus influenzae, Haemophilus aphorophilus, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus parainfluenzae , and Haemophilus paraphrophilus.
9 . The method of claim 1 wherein said rRNA target nucleic acid is from a fungus selected from the group consisting of: Histoplasma capsulatum, Blastomyces dermatitidis, Arthroderma tuberculatum, Chrysosporium keratinophilum, Coccidioides immitis, Corynascus sepedonium, Scopulariopsis acremonium, Sepedonium chrysospermum, Cryptococcus neoformans, Sacchromyces carlsbergensis, Candida albicans, Candida tropicalis, Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus terreus, Cryptococcus luteolus, Cryptococcus uniguttulatus, Oidiodendron echinulatum, Paracoccidiodes brasiliensis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus Niger, Penicillium notatum , and Malbranchea dendriticus.
10 . The method of claim 1 wherein said target nucleic acid sample undergoes amplification prior formation of said hybridization complex.
12 . The method of claim 1 wherein said electron transfer moiety is a metallocene.
13 . The method of claim 12 wherein said metallocene is a ferrocene.
14 . A method for detecting the presence of a ribosomal RNA target sequence in a nucleic acid sample, comprising:
a) applying an electronic input signal to a surface comprising an array of electrodes wherein said electrodes comprise a hybridization complex and said hybridization complex comprises:
i) a capture binding ligand;
ii) a solution binding ligand;
iii) an electron transfer moiety; and
iv) a ribosomal RNA target nucleic acid;
b) detecting the presence of said ribosomal RNA target sequence via an output signal.
15 . The method according to claim 14 wherein said electron transfer moiety is attached to said solution binding ligand.
16 . The method according to claim 14 wherein said electron transfer moiety is a metallocene.
17 . The method according to claim 16 wherein said metallocene is a ferrocene.
18 . The method of claim 14 wherein said ribosomal RNA target nucleic acid is selected from the group consisting of: 5S rRNA; 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA.
19 . The method of claim 14 wherein rRNA target nucleic acid is from a bacteria selected from the group consisting of: Mycobacterium avium, Mycobacterium intracellulare, Legionella, Salmonella, Campylobactor, Proteus mirabilis, Enterococcus, Enterobacter cloacae, Escherichia coli, Nisseria gonorrhoeae, Nisseria meningitidis, Chlamydia trachomatis, Chlamydia pssittici, Chlamydia pneumoniae, Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus hominus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulans, Staphylococcus warneri, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma Orale, Mycoplasma faucium, Mycoplasma buccale, Mycoplasma salivarium, Streptococcus pneumoniae, Streptococcus salivarius, Streptococcus agalactiae, Streptococcus bovis, Haemophilus influenzae, Haemophilus aphorophilus, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus parainfluenzae , and Haemophilus paraphrophilus.
20 . The method of claim 14 wherein said rRNA target nucleic acid is from a fungus selected from the group consisting of: Histoplasma capsulatum, Blastomyces dermatitidis, Arthroderma tuberculatum, Chrysosporium keratinophilum, Coccidioides immitis, Corynascus sepedonium, Scopulariopsis acremonium, Sepedonium chrysospermum, Cryptococcus neoformans, Sacchromyces carlsbergensis, Candida albicans, Candida tropicalis, Cryptococcus albidus, Cryptococcus laurentii, Cryptococcus terreus, Cryptococcus luteolus, Cryptococcus uniguttulatus, Oidiodendron echinulatum, Paracoccidiodes brasiliensis, Aspergillus flavus, Aspergillus fumigatus, Aspergillus Niger, Penicillium notatum , and Malbranchea dendriticus.
21 . The method of claim 14 wherein said electrodes further comprise a self assembled monolayer.
22 . The method of claim 14 wherein said electronic input signal is an AC input signal.
23 . The method of claim 14 wherein said electronic input signal is an AC/DC input signal.
24 . The method of claim 14 wherein said ribosomal RNA target nucleic acid is contacted with at least one helper probe prior to formation of said hybridization complex.
25 . The method of claim 14 wherein said solution binding ligand comprises a first portion that binds to said ribosomal RNA target nucleic acid and a second portion that comprises a recruitment linker.
26 . The method of claim 25 wherein said recruitment linker comprises at least one ETM.