Abstract: Enzyme compositions and their method of use that provide ready-to-use master mixtures. The compositions comprise a modified thermophilic DNA polymerase lacking 5?-3? and 3?-5? exonuclease activity premixed with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase and all other necessary components, including reaction buffer and nucleoside triphosphates, required to perform DNA blunting, phosphorylation, and single nucleotide extension reactions in one tube and in two steps. Among other benefits, the mixture of different enzymes, buffers and nucleoside triphosphates is stable during prolonged storage.

Abstract: Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

Abstract: A restriction endonuclease with a recognition sequence 5?-TCGA-3?. The restriction endonuclease is sensitive to the presence of a modified cytosine residue in the recognition sequence. Methods and kits using the restriction endonuclease with a recongition sequence 5?-TCGA-3? are also disclosed.

Abstract: Provided is a methylation-specific restriction endonuclease for a DNA duplex substrate, which endonuclease recognizes in a strand of the duplex a 2 to 6 nucleotide recognition sequence comprising a 5-methylcytosine, and cleaves each strand of the duplex at a fixed position outside the recognition sequence.

Abstract: Provided is a methylation-specific restriction endonuclease for a DNA duplex substrate, which endonuclease recognizes in a strand of the duplex a 2 to 6 nucleotide recognition sequence comprising a 5-methylcytosine, and cleaves each strand of the duplex at a fixed position outside the recognition sequence.

Abstract: A vector for transformation into a host cell is described comprising a toxic gene encoding a product that is lethal to the host cell, wherein the toxic gene comprises: an essential sequence region whose integrity is necessary in order for the encoded toxic gene product to be lethal to the host cell; an inessential sequence region whose integrity is not essential in order for the encoded toxic gene product to be lethal to the host cell; a regulatory sequence inserted in-frame into the inessential sequence region; and a cloning site within the essential sequence region for insertion of a nucleic acid sequence, wherein the regulatory sequence and the cloning site are positioned so as to allow the regulatory sequence to be operably linked to a nucleic acid sequence when the nucleic acid sequence is inserted into the cloning site.

Abstract: A process for producing a polynucleotide encoding a restriction endonuclease with an altered specificity, which process comprises: (a) mutagenising a polynucleotide encoding a restriction endonuclease with specificity for a recognition sequence so as to produce one or more mutated polynucleotides; and (b) isolating therefrom a polynucleotide encoding a mutated restriction endonuclease with specificity for an altered recognition sequence by selecting a polynucleotide which expresses a restriction endonuclease with methylase specificity for the altered recognition sequence.

Abstract: A strand-specific polynucleotide nickase comprising an endonuclease which comprises a first subunit and a second subunit and which recognises an asymmetric nucleotide recognition sequence, wherein the first subunit comprises a catalytic domain capable of cleaving one strand of a DNA duplex, and the second subunit is incapable of cleaving the other strand of the DNA duplex.

Abstract: A strand-specific polynucleotide nickase comprising an endonuclease which comprises a first subunit and a second subunit and which recognises an asymmetric nucleotide recognition sequence, wherein the first subunit comprises a catalytic domain capable of cleaving one strand of a DNA duplex, and the second subunit is incapable of cleaving the other strand of the DNA duplex.

Abstract: A process for producing a polynucleotide encoding a restriction endonuclease with an altered specificity, which process comprises: