The present disclosure provides, among other things, systems, methods, and kits include internal amplification controls. Provided internal amplification controls are or include non-target sequences that are amplified during nucleic acid amplification. Provided internal amplification controls are linearly amplified. Provided internal amplification controls are useful for systems, methods and kits for nucleic acid amplification.

The present disclosure provides, among other things, systems, methods, and kits include internal amplification controls. Provided internal amplification controls are or include non-target sequences that are amplified during nucleic acid amplification. Provided internal amplification controls are linearly amplified. Provided internal amplification controls are useful for systems, methods and kits for nucleic acid amplification.

Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5′ end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3′OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5′ end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) ; and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.

Methods are provided that, for example, include (a) combining ssDNA containing a modified nucleotide (e.g., a ssDNA with a modified nucleotide proximate to its 5′ end) with a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate a first ssDNA fragment having a 3′OH and a second ssDNA fragment having the modified nucleotide); wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio (m/m); and (b) cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, a method may comprise (a) combining (i) a ssDNA comprising a modified nucleotide (e.g., proximate to its 5′ end) with (ii) a DNA cleavage enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) wherein the ratio of enzyme to DNA substrate is less than 1:1 molar ratio and cleaving at least 95% of the ssDNA at the modified nucleotide. In some embodiments, methods provided herein may include (a) combining (i) a ssDNA (1) immobilized on a substrate and (2) comprising a modified nucleotide with (ii) a ssDNA cleaving enzyme capable of cleaving the ssDNA at the modified nucleotide (e.g., to generate (after cleavage) a first ssDNA fragment having a 3′OH and a second ssDNA fragment comprising the modified nucleotide) ; and (b) cleaving the immobilized ssDNA to release the second single stranded DNA fragment from the substrate. At least 95% (m/m) of an ssDNA comprising a modified nucleotide may be cleaved in less than 60 minutes.

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.

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.