Improved methods for use in nucleic acid amplification, including multiplex amplification, where the amplification is carried out in two or more distinct phases are disclosed. The first phase amplification reaction preferably lacks one or more components required for exponential amplification. The lacking component is subsequently provided in a second, third or further phase(s) of amplification, resulting in a rapid exponential amplification reaction. The multiphase protocol results in faster and more sensitive detection and lower variability at low analyte concentrations. Compositions for carrying out the claimed methods are also disclosed.

The embodiments disclosed herein utilize RNA targeting effectors to provide a robust CRISPR-based diagnostic for hemorrhagic fever virus applications. Embodiments disclosed herein can differentiate between hemorrhagic fever viruses that present with similar symptoms, as well as between strains of a hemorrhagic fever virus.

The embodiments disclosed herein utilize RNA targeting effectors to provide a robust CRISPR-based diagnostic for hemorrhagic fever virus applications. Embodiments disclosed herein can differentiate between hemorrhagic fever viruses that present with similar symptoms, as well as between strains of a hemorrhagic fever virus.

Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.

Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.

The present invention provides a method for sequencing a nucleic acid using an immersion reaction protocol. The immersion reaction protocol comprises sequentially immersing a solid support having nucleic acid molecules immobilized thereon in different reaction containers to realize nucleic acid sequencing.

The present invention provides a method for sequencing a nucleic acid using an immersion reaction protocol. The immersion reaction protocol comprises sequentially immersing a solid support having nucleic acid molecules immobilized thereon in different reaction containers to realize nucleic acid sequencing.

Methods of profiling the nucleic acid composition of single cells and tools for same. The methods can include isolating a single cell in a liquid droplet, lysing the single cell in the liquid droplet to release template nucleic acid from the cell, amplifying the template nucleic acid in the liquid droplet to generate amplified nucleic acid, and detecting the amplified nucleic acid in the liquid droplet. The methods can be useful for profiling expression patterns and/or detecting genetic characteristics such as single nucleotide polymorphisms. The tools include nucleic acid logic gates, including polymerase-dependent logic gates. The logic gates can perform logical operations such as YES, NOT, AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR, EXCLUSIVE-NOR, and IMPLY. The tools also include microfluidic systems for performing the methods.

Methods of profiling the nucleic acid composition of single cells and tools for same. The methods can include isolating a single cell in a liquid droplet, lysing the single cell in the liquid droplet to release template nucleic acid from the cell, amplifying the template nucleic acid in the liquid droplet to generate amplified nucleic acid, and detecting the amplified nucleic acid in the liquid droplet. The methods can be useful for profiling expression patterns and/or detecting genetic characteristics such as single nucleotide polymorphisms. The tools include nucleic acid logic gates, including polymerase-dependent logic gates. The logic gates can perform logical operations such as YES, NOT, AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR, EXCLUSIVE-NOR, and IMPLY. The tools also include microfluidic systems for performing the methods.

The invention provided methods and devices for performing sequential, regulated multiplex reactions in a single tube without the addition or removal of contents from the tube.