Electronically mediated nucleic acid amplification in NASBA
First Claim
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1. A method for the amplification of one or more target nucleic acids of interest in at least two samples using a bioelectronic microchip comprising:
- a) introducing the target nucleic acids of a first sample onto a bioelectronic microchip having a plurality of electronically addressable capture sites;
b) electronically addressing the target nucleic acids of the first sample to at least a first capture site which has anchored thereto at least a first oligonucleotide primer/probe comprising a capture sequence specific for one of the target nucleic acids to be amplified;
c) hybridizing the target nucleic acid of the first sample to the first oligonucleotide primer/probe at the first capture site;
d) removing any unhybridized nucleic acids of the first sample from the bioelectronic microchip,e) hybridizing target nucleic acids from at least one additional sample to the first oligonucleotide primer/probe on at least one additional capture site by, for each additional sample;
i) introducing the target nucleic acids of the additional sample onto the bioelectronic microchip;
ii) electronically addressing the target nucleic acids of the additional sample to at least one additional capture site which has anchored thereto the first oligonucleotide primer/probe, wherein the target nucleic acids of the additional sample are addressed to capture sites different than capture sites to which the first or any other additional sample'"'"'s nucleic acids are addressed;
iii) hybridizing the target nucleic acid of the additional sample to the first oligonucleotide primer/probe at the additional capture site;
iv) removing any unhybridized nucleic acids of the additional sample from the bioelectronic microchip;
f) contacting the hybridized target nucleic acids of the samples with enzymes and reagents necessary to support nucleic acid amplification, including any additional primers or probes;
g) amplifying the target nucleic acid utilizing at least one anchored amplification primer/probe to produce anchored amplicon species;
wherein the target nucleic acids from each sample are independently amplified at each capture site to which they are addressed so that the target nucleic acids from each sample are not significantly amplified at capture sites to which they are not addressed, and further wherein anchored amplicons of the same nucleic acid sequence are produced at different capture sites for target nucleic acids from at least two samples.
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Abstract
A method of improving amplification of nucleic acids using a nucleic acid sequence-based amplification (“NASBA”) method is provided wherein target nucleic acids and NASBA primers are electronically addressed to electronically addressable capture sites of a microchip. This improvement uses electronically induced hybridization of the target nucleic acids to the primers. The primers may be solution-based or immobilized on the capture sites of the microchip.
58 Citations
30 Claims
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1. A method for the amplification of one or more target nucleic acids of interest in at least two samples using a bioelectronic microchip comprising:
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a) introducing the target nucleic acids of a first sample onto a bioelectronic microchip having a plurality of electronically addressable capture sites; b) electronically addressing the target nucleic acids of the first sample to at least a first capture site which has anchored thereto at least a first oligonucleotide primer/probe comprising a capture sequence specific for one of the target nucleic acids to be amplified; c) hybridizing the target nucleic acid of the first sample to the first oligonucleotide primer/probe at the first capture site; d) removing any unhybridized nucleic acids of the first sample from the bioelectronic microchip, e) hybridizing target nucleic acids from at least one additional sample to the first oligonucleotide primer/probe on at least one additional capture site by, for each additional sample; i) introducing the target nucleic acids of the additional sample onto the bioelectronic microchip; ii) electronically addressing the target nucleic acids of the additional sample to at least one additional capture site which has anchored thereto the first oligonucleotide primer/probe, wherein the target nucleic acids of the additional sample are addressed to capture sites different than capture sites to which the first or any other additional sample'"'"'s nucleic acids are addressed; iii) hybridizing the target nucleic acid of the additional sample to the first oligonucleotide primer/probe at the additional capture site; iv) removing any unhybridized nucleic acids of the additional sample from the bioelectronic microchip; f) contacting the hybridized target nucleic acids of the samples with enzymes and reagents necessary to support nucleic acid amplification, including any additional primers or probes; g) amplifying the target nucleic acid utilizing at least one anchored amplification primer/probe to produce anchored amplicon species; wherein the target nucleic acids from each sample are independently amplified at each capture site to which they are addressed so that the target nucleic acids from each sample are not significantly amplified at capture sites to which they are not addressed, and further wherein anchored amplicons of the same nucleic acid sequence are produced at different capture sites for target nucleic acids from at least two samples. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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Specification