The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches. Also provided are novel nucleic acid sequences encoding mismatch endonucleases, polypeptides encoded thereby, as well as nucleic acid constructs, transgenic cells, and various compositions thereof.

The present invention relates to the analysis of complex single cell sequencing libraries. Disclosed are methods for enrichment of library members based on the presence of cell-of origin barcodes to identify and concentrate DNA that is relevant to interesting cells or components that would be expensive or difficult to study otherwise. Also, disclosed are methods of capturing cDNA library molecules by use of CRISPR systems, hybridization or PCR. The present invention allows for identifying the properties of rare cells in single cell RNA-seq data and accurately profile them through clustering approaches. Further information on transcript abundances from subpopulations of single cells can be analyzed at a lower sequencing effort. The methods also allow for linking TCR alpha and beta chains at the single cell level.

The present disclosure describes methods and microfluidic devices for generating clusters of amplicons for a nucleic acid library, and their uses for high-throughput DNA sequencing or detection of target polynucleotides in a sample.

The present invention provides materials and methods useful for error correction of nucleic acid molecules. In one embodiment of the invention, a first plurality of double-stranded nucleic acid molecules having a nucleotide mismatch are fragmented by exposure to a molecule having unidirectional mismatch endonuclease activity. The nucleic acid molecules are cut at the mismatch site or near the mismatch site, leaving a double-stranded nucleic acid molecule having a mismatch at the end or near end of the molecule. The nucleic acid molecule is then exposed to a molecule having unidirectional exonuclease activity to remove the mismatched nucleotide. The missing nucleotides can then be filled in by the action of, e.g., a molecule having DNA polymerase activity. The result is double-stranded nucleic acid molecules with a decreased frequency of nucleotide mismatches. Also provided are novel nucleic acid sequences encoding mismatch endonucleases, polypeptides encoded thereby, as well as nucleic acid constructs, transgenic cells, and various compositions thereof.

Purified Ref polypeptides with increased nuclease site-specific targeting activity, recombinant nucleic acids and cells for expression of such Ref polypeptides, and methods for using the Ref polypeptides in combination with RecA protein and variants thereof to effect targeted nuclease cleavage of a DNA duplex are disclosed.

Purified Ref polypeptides with increased nuclease site-specific targeting activity, recombinant nucleic acids and cells for expression of such Ref polypeptides, and methods for using the Ref polypeptides in combination with RecA protein and variants thereof to effect targeted nuclease cleavage of a DNA duplex are disclosed.

Purified Ref polypeptides with increased nuclease site-specific targeting activity, recombinant nucleic acids and cells for expression of such Ref polypeptides, and methods for using the Ref polypeptides in combination with RecA protein and variants thereof to effect targeted nuclease cleavage of a DNA duplex are disclosed.

Provided herein are fusion proteins and associated methods and systems for increasing the efficiency of genome editing using site-directed nucleases. The fusion proteins, systems, and methods can selectively increase desired editing outcomes (e.g., inversion, excision, and homology-directed repair). Also provided are various useful compositions for the production and use of the fusion proteins and practice of the methods.

Provided are a method for preparing a linear closed DNA and a plasmid for use in the method. The plasmid comprises two editable regions of prokaryotic telomerase target sequences which are connected in tandem in the same direction, and both ends of the editing regions are independently provided with one or more restriction endonuclease digestion sites. The method can yield high-purity LcDNA and can be better applied to clinical research and commercial applications.

The present invention relates to enzymes having DNA polymerase and 3-5 exonuclease activities. In particular, the present invention relates to a heat labile enzyme possessing a DNA polymerase II activity and a 3-5 exonuclease activity of marine origin. Furthermore, the present invention relates to a DNA polymerase primarily exerting a 3-5 activity, i.e. where the polymerase activity is absent. The present invention furthermore relates to the use of the exonuclease activity to degrade the 3-5 strand of double stranded DNA to perform single stranded overhang, e.g. in recombinant cloning processes, or in processes for removal of contaminating nucleic acid molecules.