Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

This invention relates to methods and compositions for amplifying nucleic acids, e.g., genomic DNA, using nicking agents. The method of amplifying nucleic acids comprising: (a) forming a reaction mixture comprising: (i) a first nucleic acid template comprising a strand having a first nicking agent recognition sequence; (ii) a second nucleic acid template comprising a strand having a second nicking agent recognition sequence; (iii) at least one primer for a target region on the first or second nucleic acid template; (iv) at least one protein having DNA polymerase domain function, wherein the domain function comprises a first domain function capable of strand displacement activity and a second domain function capable of high processivity activity, or at least one protein having DNA polymerase domain function capable of strand displacement activity and at least one protein having DNA polymerase domain function capable of high processivity activity; (v) at least one deoxynucleoside triphosphate; and (vi) a first nicking agent for recognizing the first nicking agent recognition sequence and a second nicking agent for recognizing the second nicking agent recognition sequence; (b) incubating the reaction mixture under conditions that amplifies the nucleic acid templates, wherein the domain functions capable of strand displacement activity and high processivity activity are separate from each other and capable of carrying out their activities simultaneously. In specific embodiments, the nicking agent is NB.BsrDI and the proteins having DNA polymerase domain functions are Bst 3.0 polymerase and Pfu polymerase.

This invention relates to methods and compositions for amplifying nucleic acids, e.g., genomic DNA, using nicking agents. The method of amplifying nucleic acids comprising: (a) forming a reaction mixture comprising: (i) a first nucleic acid template comprising a strand having a first nicking agent recognition sequence; (ii) a second nucleic acid template comprising a strand having a second nicking agent recognition sequence; (iii) at least one primer for a target region on the first or second nucleic acid template; (iv) at least one protein having DNA polymerase domain function, wherein the domain function comprises a first domain function capable of strand displacement activity and a second domain function capable of high processivity activity, or at least one protein having DNA polymerase domain function capable of strand displacement activity and at least one protein having DNA polymerase domain function capable of high processivity activity; (v) at least one deoxynucleoside triphosphate; and (vi) a first nicking agent for recognizing the first nicking agent recognition sequence and a second nicking agent for recognizing the second nicking agent recognition sequence; (b) incubating the reaction mixture under conditions that amplifies the nucleic acid templates, wherein the domain functions capable of strand displacement activity and high processivity activity are separate from each other and capable of carrying out their activities simultaneously. In specific embodiments, the nicking agent is NB.BsrDI and the proteins having DNA polymerase domain functions are Bst 3.0 polymerase and Pfu polymerase.

The present invention relates to a method for producing a panel of cells (i.e. a cell library) expressing various different mutant variants of a protein of interest, wherein only one of said mutant variants is expressed per cell from a single gene copy. The present invention also relates to a method or cell library for identifying a mutant variant of a protein of interest having a different or modified biological activity as compared to the corresponding wild-type protein of interest. According to the present invention the identified mutant variant of a protein of interest may be applied for white biotechnology.

The present invention relates to a method for producing a panel of cells (i.e. a cell library) expressing various different mutant variants of a protein of interest, wherein only one of said mutant variants is expressed per cell from a single gene copy. The present invention also relates to a method or cell library for identifying a mutant variant of a protein of interest having a different or modified biological activity as compared to the corresponding wild-type protein of interest. According to the present invention the identified mutant variant of a protein of interest may be applied for white biotechnology.

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.

Compositions, methods and kits are provided for identifying the presence and location of a target in chromosomal DNA. A nicking endonuclease fused to a binding domain that binds to a constant region of an antibody (NEFP) is provided that may be used for binding to a target directly or via an antibody that binds to the target. The target may be a protein or structural feature of the DNA and its presence and location may correspond to a phenotype and/or pathology in a biopsy or other cell sample for diagnostic purposes. The background is reduced by the addition of a glycoaminoglycan (GAG) that reversibly inhibits binding of the NEFP to DNA. Nick translation in the presence of a strand displacing polymerase enables the incorporation of tagged nucleotides that (i) blocks re-nicking; (ii) facilitates immobilization of DNA fragments around the target for sequencing; and/or (iii) enables dye labelling of the chromosomal DNA within the cell nuclei for analysis by microscopy.