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.

In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.

Provided herein are methods and compositions to determine the efficacy of a nucleic acid pre-amplification reaction.

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.

Compositions and methods for highly specific nucleic acid probes and primers are provided. The probe system comprises a complement strand and a protector stand that form a partially double-stranded probe. The reaction standard free energy of hybridization between the probe and target nucleic acid as determined by Expression 1 (ΔG°.sub.rxn=ΔG°.sub.t-TC−ΔG°.sub.nh-PC+(ΔG°.sub.v-TC−ΔG°.sub.h-PC)) is from about −4 kcal/mol to about +4 kcal/mol. Alternatively, the reaction standard free energy of hybridization between the probe and target nucleic acid is determined by Expression 1 to be within 5 kcal/mol of the standard free energy as determined by Expression 2 (−Rτln(([P].sub.0−[C].sub.0)/[C].sub.0)]), where the [P].sub.0 term of Expression 2 equals the concentration of the protector strand and the [C].sub.0 term of Expression 2 equals the concentration of the complement strand. In addition, a method for on-the-fly fine tuning of a reaction using the present probe is provided.

Compositions and methods for highly specific nucleic acid probes and primers are provided. The probe system comprises a complement strand and a protector stand that form a partially double-stranded probe. The reaction standard free energy of hybridization between the probe and target nucleic acid as determined by Expression 1 (ΔG°.sub.rxn=ΔG°.sub.t-TC−ΔG°.sub.nh-PC+(ΔG°.sub.v-TC−ΔG°.sub.h-PC)) is from about −4 kcal/mol to about +4 kcal/mol. Alternatively, the reaction standard free energy of hybridization between the probe and target nucleic acid is determined by Expression 1 to be within 5 kcal/mol of the standard free energy as determined by Expression 2 (−Rτln(([P].sub.0−[C].sub.0)/[C].sub.0)]), where the [P].sub.0 term of Expression 2 equals the concentration of the protector strand and the [C].sub.0 term of Expression 2 equals the concentration of the complement strand. In addition, a method for on-the-fly fine tuning of a reaction using the present probe is provided.

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.

The present application relates to methods for detecting a first allele of a single nucleotide polymorphism (SNP) in a nucleic acid sequence under isothermal conditions using primers specific for said first allele, in particular using Loop mediated isothermal amplification (LAMP), wherein the amplification of a second allele is prevented by using blocking primers.