Disclosed is a method for amplifying and detecting polynucleotides which can provide sensitive, specific detection of multiple targets from a clinical specimen within a relatively short time.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.

The present invention addresses the issue of providing a target base sequence detection method, etc., whereby a determination can be readily made regarding whether or not a target base sequence is present in a nucleic acid sample. A fluorescent-labeled detection probe and a competitive probe are added to a nucleic acid sample and caused to hybridize with the nucleic acid in the sample, the fluorescence intensity is measured while changing the temperature of the reaction sample, and first order differentiation is performed on a temperature-fluorescence intensity curve. The fluorescent-labeled detection probe and competitive probe base length, base sequence, and amount to be added to nucleic acid samples are determined such that the first order derivative curve for a control target reaction sample including a target base sequence has a peak but the first order derivative curve for a control non-target reaction sample including a non-target base sequence does not substantially have a peak, when: the fluorescent-labeled detection probe and the competitive probe are added to both the control target nucleic acid sample and the control non-target nucleic acid sample; the fluorescence intensity is measured while the temperature of both the obtained control target reaction sample and the control non-target reaction sample are changed; and first order differentiation is performed on a temperature-fluorescence intensity curve.

Methods and reagent for determining the presence and/or for quantifying the amount of a target nucleic acid sequences in a sample are provided. In some aspects, the methods comprise performing a melt analysis by detecting, a signal from a probe at a temperature that is lower than the Tm of the probe and a signal at a temperature that is higher than the Tm of the probe, without detecting a signal at the Tm of the probe.

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.

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.

Provided herein are reagents for improving PCR accuracy.