Provided herein are reagents for improving PCR accuracy.

Methods, devices, and kits are provided for performing PCR in <20 seconds per cycle, with improved efficiency and yield.

Methods, devices, and kits are provided for performing PCR in <20 seconds per cycle, with improved efficiency and yield.

Provided herein are methods for rapid detection of RNA in a sample. The methods comprise providing a reaction mixture containing the sample, amplification reagents, and a polymerase enzyme having both RNA and DNA-dependent polymerase activity; reverse transcribing the RNA to DNA by incubating for a reverse transcription time of no longer than 5 minutes; and amplifying the DNA by performing a thermal cycling protocol comprising a plurality of amplification cycles, wherein each amplification cycle comprises at least a denaturation step and an annealing step.

Provided herein are methods for rapid detection of RNA in a sample. The methods comprise providing a reaction mixture containing the sample, amplification reagents, and a polymerase enzyme having both RNA and DNA-dependent polymerase activity; reverse transcribing the RNA to DNA by incubating for a reverse transcription time of no longer than 5 minutes; and amplifying the DNA by performing a thermal cycling protocol comprising a plurality of amplification cycles, wherein each amplification cycle comprises at least a denaturation step and an annealing step.

Provided is a method, including hybridizing test primers to immobilized primers, wherein the immobilized primers include a predetermined sequence of nucleotides and are attached to a substrate through their 5-prime ends, individual test primers are complementary to a portion of each of at least some of the immobilized primers, and no more than one test primer molecule hybridizes to an immobilized primer molecule, extending, by only one nucleotide, at least some of the test primers with a polymerase according to templates, wherein said templates comprise immobilized primers hybridized to said test primers and nucleotides incorporated into extended test primers comprise a fluorescent tag, and detecting an amount of fluorescent test primers.

Provided is a method, including hybridizing test primers to immobilized primers, wherein the immobilized primers include a predetermined sequence of nucleotides and are attached to a substrate through their 5-prime ends, individual test primers are complementary to a portion of each of at least some of the immobilized primers, and no more than one test primer molecule hybridizes to an immobilized primer molecule, extending, by only one nucleotide, at least some of the test primers with a polymerase according to templates, wherein said templates comprise immobilized primers hybridized to said test primers and nucleotides incorporated into extended test primers comprise a fluorescent tag, and detecting an amount of fluorescent test primers.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

The present disclosure provides methods for determining the ploidy status of a chromosome in a gestating fetus from genotypic data measured from a mixed sample of DNA comprising DNA from both the mother of the fetus and from the fetus, and optionally from genotypic data from the mother and father. The ploidy state is determined by using a joint distribution model to create a plurality of expected allele distributions for different possible fetal ploidy states given the parental genotypic data, and comparing the expected allelic distributions to the pattern of measured allelic distributions measured in the mixed sample, and choosing the ploidy state whose expected allelic distribution pattern most closely matches the observed allelic distribution pattern. The mixed sample of DNA may be preferentially enriched at a plurality of polymorphic loci in a way that minimizes the allelic bias, for example using massively multiplexed targeted PCR.

Arrays of single molecules and methods of producing an array of single molecules are described. Arrays with defined volumes between 10 attoliters and 50 picoliters enable single molecule detection and quantitation.