High density sequence detection methods and apparatus
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Accused Products
Abstract
Methods for amplifying polynucleotides, e.g., by PCR, in a sample comprising polynucleotide targets present at very low concentration, comprising: (a) applying amplification reactants to the surface of a substrate comprising reaction spots, wherein the reactants comprise the sample and an amplification reagent; (b) forming a sealed reaction chamber, having a volume less than about 120 nanoliters, preferably less than about 20 nanoliters, over each of said reaction spots; and (c) thermal cycling the substrate and reactants. In one embodiment, the forming step comprises loading a sealing fluid, e.g., mineral oil, on the surface so as to cover the reaction spots. The present invention also provides microplates, comprising: (a) a substrate having at least about 10,000 reaction spots, each comprising a primer and a droplet of reagent having a volume less than about 120 nanoliters, preferably less then about 20 nanoliters; and (b) a sealing liquid isolating each of the spots.
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Citations
118 Claims
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1-90. -90. (canceled)
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91. A method for performing PCR on a liquid sample comprising a plurality of polynucleotide targets, each polynucleotide target being present at very low concentration within the sample, comprising:
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applying PCR reactants to the surface of a substrate to produce a plurality of reaction spots on the surface of the substrate;
loading the liquid sample and a PCR reagent mixture onto the reaction spots;
forming a sealed reaction chamber, having a volume of less than about 20 nanoliters, over each of the reaction spots; and
amplifying the sample. - View Dependent Claims (92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)
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103. A method for simultaneously quantitatively detecting a plurality of polynucleotide targets in a liquid sample comprising a genomic mixture of polynucleotides present at very low concentration, comprising:
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(a) distributing the liquid sample into an array of reaction chambers on a planar substrate, wherein (i) each chamber has a volume of less than about 100 nanoliters, and (ii) each chamber comprises (1) at least one amplification primer for one of the polynucleotide targets, and (2) a probe associated with the primer which emits a concentration dependent signal if the amplification primer binds with a polynucleotide, and (iii) the array comprises at least one chamber comprising at least one amplification primer for each of the polynucleotide targets;
(b) performing amplification on the samples in the array so as to increase the concentration of polynucleotide in each of the chambers in which the polynucleotide binds to a amplification primer; and
(c) identifying which of the reaction chambers contains a polynucleotide that has been bound to a amplification primer, by detecting the presence of the probe associated with the amplification primer. - View Dependent Claims (104, 105, 106, 107, 108, 109, 110)
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111. A microplate, for use in performing amplification by PCR on a liquid sample comprising a plurality of polynucleotide targets, comprising:
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(a) a substrate having at least about 10,000 reaction spots, each spot comprising a primer set, a probe set and an amplification reagent having a volume of less than about 20 nanoliters; and
(b) a sealing liquid covering said substrate and isolating each of said reaction spots. - View Dependent Claims (112, 113, 114, 115, 116, 117, 118)
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Specification