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.

Disclosed herein is a nucleic acid amplification process referred to as Linear-Expo-Linear Polymerase Chain Reaction (LEL-PCR).

Disclosed herein is a nucleic acid amplification process referred to as Linear-Expo-Linear Polymerase Chain Reaction (LEL-PCR).

The present disclosure generally relates to nucleic acid amplification systems and methods suitable for construction of nucleic acid samples, including construction of normalized nucleic acid libraries. In some embodiments, the method includes providing one or more input nucleic acid samples, contacting each of the input nucleic acid samples (e.g., input library) with a reaction mixture including first amplification or normalization primers and second amplification or normalization primers, wherein the first amplification or normalization primers are immobilized on a solid support and the second amplification or normalization primers are in solution phase, and amplifying the input nucleic acid samples under conditions such that substantially all of the first amplification or normalization primers are incorporated into amplification products. Further provided are systems and droplet actuator devices that are configured to carry out the methods disclosed herein. Compositions that include nucleic acid samples and libraries, preferably normalized, prepared in accordance with the disclosed methods and systems are also provided.

The present disclosure generally relates to nucleic acid amplification systems and methods suitable for construction of nucleic acid samples, including construction of normalized nucleic acid libraries. In some embodiments, the method includes providing one or more input nucleic acid samples, contacting each of the input nucleic acid samples (e.g., input library) with a reaction mixture including first amplification or normalization primers and second amplification or normalization primers, wherein the first amplification or normalization primers are immobilized on a solid support and the second amplification or normalization primers are in solution phase, and amplifying the input nucleic acid samples under conditions such that substantially all of the first amplification or normalization primers are incorporated into amplification products. Further provided are systems and droplet actuator devices that are configured to carry out the methods disclosed herein. Compositions that include nucleic acid samples and libraries, preferably normalized, prepared in accordance with the disclosed methods and systems are also provided.

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 invention provides methods of amplification from a single primer or a pair of forward and reverse primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid.

A nucleic acid amplification reaction method includes subjecting a reaction mixture containing a nucleic acid amplification reaction reagent to be used for amplifying a nucleic acid to a thermal cycle for amplifying the nucleic acid, wherein in the thermal cycle, a heating time for an annealing reaction and an elongation reaction is 1 sec or more and 10 sec or less, the nucleic acid amplification reaction reagent contains a forward primer, a reverse primer, a polymerase, and a fluorescently labeled probe, the concentration of the forward primer is 0.4 μM or more and 3.2 μM or less, the concentration of the reverse primer is 0.4 μM or more and 3.2 μM or less, the amount of the polymerase is 0.5 U or more and 4 U or less, and the concentration of the fluorescently labeled probe is 0.15 μM or more and 1.2 μM or less.

A nucleic acid amplification reaction method includes subjecting a reaction mixture containing a nucleic acid amplification reaction reagent to be used for amplifying a nucleic acid to a thermal cycle for amplifying the nucleic acid, wherein in the thermal cycle, a heating time for an annealing reaction and an elongation reaction is 1 sec or more and 10 sec or less, the nucleic acid amplification reaction reagent contains a forward primer, a reverse primer, a polymerase, and a fluorescently labeled probe, the concentration of the forward primer is 0.4 μM or more and 3.2 μM or less, the concentration of the reverse primer is 0.4 μM or more and 3.2 μM or less, the amount of the polymerase is 0.5 U or more and 4 U or less, and the concentration of the fluorescently labeled probe is 0.15 μM or more and 1.2 μM or less.

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.