Presented herein are altered polymerase enzymes for improved incorporation of nucleotides and nucleotide analogues, in particular altered polymerases that maintain low pre-phasing rates when using ambiently stored polymerases, as well as methods and kits using the same.

The present invention refers to a fusion protein or a protein complex comprising a DNA binding protein (DnaBP), a nucleobase modifying protein (NMP), and a Base Excision Repair associated protein (BERAP. Also, described herein are a method of replacing a cytosine with a guanine on a DNA strand in a cell and a method of treating a subject having or suspected of having a disease or disorder.

The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Provided are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of primer extension methods. Also provided are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

The present invention concerns reagents for the reversible protection of biological molecules. It relates in particular to compounds derived from azaisatoic anhydride and their uses for the protection of biological molecules, particularly enzymes, in order to block their activity. The invention also relates to the biological molecules protected in this manner and to the methods for making use of these reagents.

Recombinant DP04-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DP04 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

The invention relates to the field of nucleic acid synthesis or sequencing, more specifically to methods for ab-initio synthesis of nucleic acids, comprising contacting a nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3′-hydroxyl group of the nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of both ab-initio single-stranded nucleic acid synthesis activity and template-independent terminal nucleotidyl transferase activity.

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, extreme thermostable single-stranded DNA binding protein (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.

This disclosure provides an E. coli strain, which lacks thioredoxin reductase activity encoded by trxB and thioredoxin 1 activity encoded by trxA, and glutathione reductase activity encoded by gor. Said E. coli strain expresses a mutated AhpC protein having glutathione reductase activity and a cytosolic prokaryotic disulfide isomerase. The E. coli strain has an oxidative cytosol and can be used to efficiently produce proteins having disulfide bonds.