| 摘要 |
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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