The disclosure provides a high-fidelity polymerase with preference for gapped DNA and use thereof. The Klenow fragment (KlenDr) derived from Deinococcus radiodurans DNA polymerase I, which has the high-fidelity polymerization characteristics, is independent of 3′-5′ proofreading exonuclease activity, has the preference for binding gapped DNA, and is different from the existing commercial high-fidelity polymerase. Due to the specific affinity of KlenDr to gapped DNA substrate, the 3′ end of the forward primer will not be cut off, and the downstream nucleotide chain is rarely replaced.

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 relates, e.g., to in vitro method, using isolated protein reagents, for joining two double stranded (ds) DNA molecules of interest, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule share a region of sequence identity, comprising contacting the two DNA molecules in a reaction mixture with (a) a non-processive 5′ exonuclease; (b) a single stranded DNA binding protein (SSB) which accelerates nucleic acid annealing; (c) a non strand-displacing DNA polymerase; and (d) a ligase, under conditions effective to join the two DNA molecules to form an intact double stranded DNA molecule, in which a single copy of the region of sequence identity is retained. The method allows the joining of a number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes.

Provided are a modified Klenow fragment and an application thereof; specifically provided is a modified Klenow fragment, wherein at least one position or functionally equivalent position among F762, A842, I709 and P603 in the amino acid sequence of the modified Klenow fragment contains at least one amino acid substitution mutation. The modified Klenow fragment has higher DNA polymerase activity than a wild-type Klenow fragment and can be applied to sequencing.

Provided herein are engineered variants of archaeal, prokaryotic, and eukaryotic polymerases that exhibit enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, and improved uracil-tolerance, in polymerase-catalyzed nucleotide extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases, forming binding complexes and forming ternary complexes, and uses for conducting nucleic acid sequencing reactions.

Recombinant DPO4-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 accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

The disclosure provides systems, methods, and compositions for a target specific nuclease and a blunting enzyme to correct frameshift mutations for genome editing and treatment of diseases. In some embodiments, the target specific nuclease and the blunting enzyme are combined with a guide RNA and/or a microhomology-mediated end joining (MMEJ) inhibitor.

Disclosed are methods for isolating polymerase complexes from a mixture of polymerase complex components. The polymerase complexes can comprise a nanopore to provide isolated nanopore sequencing complexes. The methods relate to the positive and negative isolation of the polymerase complexes and/or nanopore sequencing complexes. Also disclosed is a nucleic acid adaptor for isolating active polymerase complexes, polymerase complexes comprising the nucleic acid adaptor, and methods for isolating active polymerase complexes using the nucleic acid adaptor.

The present disclosure includes compositions and methods for improved DNA amplification reactions. In particular, the present disclosure provides compositions and methods for hot-start PCR applications using DNA polymerase inhibitors that minimize non-specific DNA amplification by inactivating DNA polymerase at lower temperatures.

A method for purifying nucleotides is provided, that includes preparing a solution comprising (a) 3′-blocked nucleotides, (b) 3′-OH nucleotides, (c) a polishing polymerase, and (d) a template. The polishing polymerase and the template are used to selectively polymerize the 3′-OH nucleotides and thus reduce a concentration in the solution of the 3′-OH nucleotides relative to the 3′-blocked nucleotides.