Method for nucleic acid detection using voltage enhancement
First Claim
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1. A method of discrete analysis of individual nucleic acid constructs in an array, said method comprising:
- (a) providing a device comprising a substrate comprising a surface and a plurality of nucleic acid macromolecules disposed on the surface at localized discrete attachment sites;
(b) hybridizing a probe of known sequence having an optically detectable label to each of at least some of the nucleic acid macromolecules under conditions that permit formation of perfectly matched duplexes between the probe and complementary sequences in the nucleic acid macromolecules;
(c) applying a positive charge to the substrate surface to promote compacting of the macromolecules for discrete optical detection; and
(d) optically detecting the labeled duplexes while the positive voltage is applied, thereby identifying a nucleotide sequence in at least some of the nucleic acid macromolecules.
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Abstract
Methods are provided for carrying out DNA sequencing on a device having upper and lower conductive layers separated by an insulative layer. Holes in the upper conductive layer create discrete attachment sites for DNA fragments. Voltage is applied to the surface to control affinity between the attachment sites and the DNA fragments, and to compact the DNA fragments for discrete optical detection.
113 Citations
24 Claims
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1. A method of discrete analysis of individual nucleic acid constructs in an array, said method comprising:
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(a) providing a device comprising a substrate comprising a surface and a plurality of nucleic acid macromolecules disposed on the surface at localized discrete attachment sites; (b) hybridizing a probe of known sequence having an optically detectable label to each of at least some of the nucleic acid macromolecules under conditions that permit formation of perfectly matched duplexes between the probe and complementary sequences in the nucleic acid macromolecules; (c) applying a positive charge to the substrate surface to promote compacting of the macromolecules for discrete optical detection; and (d) optically detecting the labeled duplexes while the positive voltage is applied, thereby identifying a nucleotide sequence in at least some of the nucleic acid macromolecules. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of preparing nucleic acid macromolecules in an array, said method comprising:
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providing a device comprising a substrate comprising a surface and a plurality of nucleic acid macromolecules to be analyzed singly disposed on the surface at localized discrete attachment sites, said substrate surface having a positive electric charge at the discrete attachment sites; reducing but not removing the positive charge on the discrete attachment sites so as to relax the attached macromolecules in preparation for hybridizing probes of known sequence to the nucleic acid macromolecules at the attachment sites; and hybridizing the probes to the nucleic acid macromolecules under conditions that permit formation of perfectly matched duplexes between the probes and complementary sequences in the nucleic acid macromolecules; and compacting the nucleic acid molecules by increasing the positive charge on the discrete attachment sites after hybridizing the probes, wherein hybridization of each probe is indicative of a sequence in the nucleic acid macromolecule to which it is hybridized. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
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20. A method of compacting individual nucleic acid constructs in an array for discrete analysis, said method comprising:
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providing a device comprising a plurality of different nucleic acid macromolecules to be analyzed that are singly disposed on a substrate surface at localized discrete attachment sites that are separated by a distance of 0.3 μ
m to 10 μ
m;hybridizing labeled probes of known sequence to the nucleic acid macromolecules under conditions that permit formation of perfectly matched duplexes between the probes and complementary sequences in the nucleic acid macromolecules, wherein hybridization of the probe is indicative of a sequence in the nucleic acid macromolecule(s) to which it is hybridized; applying a positive voltage to the substrate surface after hybridizing the probes such that the nucleic acid macromolecules are compacted for discrete analysis. - View Dependent Claims (21, 22, 23, 24)
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Specification