Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain high fidelity under reduced incorporation times, as well as methods and kits using the same.

The present invention provides a heat-resistant DNA polymerase mutant with high amplification activity. Particularly, the present invention uses protein directed evolution technology to construct a random mutation library for the polymerase active domain of Taq enzyme, and gradually adds screening pressure, so that unsuitable mutations will be eliminated naturally, and mutations with dominant traits will gradually accumulate. Finally, a series of amino acid sites and their mutations that are critical to Taq enzyme amplification and polymerization performance will be selected, and a Taq enzyme mutant with high amplification activity will be obtained.

Disclosed is a preparation method for a DNA next-generation sequencing library, including steps: digestion, end repair, and A-tailing of genomic DNA; adapter ligation of DNA fragments; bead purification of product after adapter ligation; PCR amplification of DNA fragments; and selection and purification of PCR product fragments. The preparation method for the DNA next-generation sequencing library includes steps: fractionating by VVN and T7 through a single-step reaction process with double digestion and end repair, blunting a 5′-overhang under the polymerization action of a Taq DNA polymerase, adding an A (adenine) to a 3′-end, and achieving the preparation of the DNA next-generation sequencing library under an integrated single-step reaction. After the single-step reaction ends, bead purification isn't required, so that the preparation process is simple. In the preparation process, there is no preference for two restriction enzymes, which achieves the sequencing of target fragments.

The present invention relates to signalling catalytic nucleic acid nanostructures that are responsive to polymerase activity, methods of their use, devices and kits comprising the same. More specifically, the present invention provides a catalytic signalling nanostructure comprising a DNAzyme/RNAzyme, such as G-quadruplex hemin, and a polymerase—responsive element. Polymerase elongation of the polymerase-responsive element eliminates catalytic activity of the DNAzyme/RNAzyme. The catalytic nucleic acid nanostructure can be used alone or paired with a target recognition nanostructure which can transduce molecular signals into polymerase activity, in an integrated circuit.

Provided is a method for detecting an FNA virus in a biological sample, the method comprising: (1) a step for preparing a sample solution containing a biological sample, a protease, a nucleic acid that does not become a target of nucleic acid amplification, and at least one additive selected from the group consisting of chaotropic reagents and surfactants; (2) a step for preparing a nucleic acid amplification reaction solution containing the sample solution prepared in step (1) and containing a polypeptide having reverse transcriptase activity and DMA polymerase activity or a polypeptide having reverse transcription activity and a polypeptide having DNA polymerase activity; and (3) a step for amplifying a nucleic acid of the RNA virus in the reaction solution prepared in step (2).

The present invention features methods for manufacturing an electrochemical sensor for detecting a diagnostic target oligonucleotide. The methods described herein provide for an electrochemical sensor with a higher level of coverage of the probes on its surface, thus allowing for more sensitive detection of a target oligonucleotide. The methods may feature first mixing disulfide terminated oligonucleotides having a free thiol moiety at the 3′ end with a gold substrate and subsequently introducing to the gold substrate a composition for reducing thiol moieties to cause the oligonucleotides to bind to the surface of the gold substrate. In some embodiments, the method comprises removing excess thiol and oligonucleotides, which may help with non-competitive binding. In some embodiments, the method comprises rinsing the gold substrate with water and drying with nitrogen.

Provided are HBV immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors expressing such immunogenic polypeptides for use in eliciting an immune response against HBV; pharmaceutical and immunogenic compositions and kits comprising such polypeptides, polynucleotides or vectors, and methods of use in treating and/or preventing HBV.

Methods, kits and compositions, in some embodiments, may include a thermostable DNA guided Argonaute protein for example TtAgo, a thermostable single-stranded DNA binding protein (SSB) for example, ET SSB, and, optionally, a strand-displacing polymerase. A SSB may allow (a) Argonaute/guide DNA complexes to substantially enhance cleavage efficiency of single- and double-stranded DNA substrates; (b) the use of longer guide DNAs (e.g., guide DNAs that are at least 24 nucleotides in length) and/or (c) increases in the sequence specificity of Argonaute-mediated binding and cleavage reactions.

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Disclosed herein is a specific B-family DNA polymerase variants that exhibit an improved incorporation of nucleotide analogues for synthesizing polynucleotides and sequencing the associated nucleic acid template. More particularly, the DNA sequencing-by-synthesis method can be efficiently performed by said B-family DNA polymerase variants with a normal nucleic acid template and reversible dye-terminator nucleotides to precisely determine the sequence of associated nucleic acid template.