Thermo Fisher Scientific Baltics Uab

Thermo Fisher Scientific Gmbh

Remigijus Skirgaila

Arvydas Janulaitis

Dangira Siksniene

Renata Rimseliene

Mindaugas Ukanis

Rasa Sukackaite

Skaiste Valinskyte

Martyna Simutyte

Karolis Matjosaitis

Sigitas Burinskas

Tadas Povilaitis

Agne Tubeleviciūté

Renata Rims̆eliēnē

Renata Rimseliėnė

Renata Rimselienė

Arvydas Lubys

This disclosure relates to thermophilic family B DNA polymerases comprising a neutral amino acid residue at a certain position near the C-terminus of the catalytic domain, which corresponds to a position occupied by a basic amino acid residue in wild-type Pfu polymerase. Related uses, methods, and compositions are also provided. In some embodiments, the polymerases comprise a 3'-5&#x2032; exonuclease domain and/or a sequence non-specific dsDNA binding domain.

Thermophilic DNA polymerase mutants

A DNA polymerase mutant comprising a Taq DNA polymerase amino acid sequence with a mutation at one or more of the following selected amino acid positions: E189K, E230K, E507K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, F749V; wherein the polymerase mutant exhibits relative to wild-type DNA polymerase increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor; and wherein, when the mutation is E507K in combination with two or more further mutations or the mutation is Q680R in combination with four or more further mutations, at least one of the further mutations is at one of the selected amino acid positions; and when the mutation is I553V, this is not in combination with D551S.

DNA polymerases

A method for the production of nucleic acid encoding a target protein. The method comprises (a) providing an array of RNA or DNA molecules including one or more encoding the target protein; (b) generating a target protein from the array to form RNA-protein or DNA-protein complexes in which the RNA or DNA molecule is non-covalently or covalently bound to the complex; (c) separating the complexes into compartments wherein most or all of the compartments contain no more than one complex; (d) subjecting the complexes to reaction conditions which allow target protein activity; and (e) selecting nucleic acid encoding the target protein on the basis of the activity associated therewith, wherein when the complex is a DNA-protein complex in which the DNA is non-covalently bound, step b) is performed in the absence of separate compartments for each complex.

Production of nucleic acid

Mutants of bacteriophage phi29 DNA polymerase with increased protein stability and increased half-life, compared to wild type DNA polymerase. The disclosed mutants are more stable in reaction mixtures with or without DNA. The inventive phi29 DNA polymerase mutants generate more amplification product. The inventive phi29 DNA polymerase mutants amplify genomic DNA with less bias compared to wild type DNA polymerase. Selected mutations increase the affinity of polymerase for DNA template.

Phi29 DNA polymerase mutants having increased thermostability and processivity

The invention provides novel mutant thermostable DNA polymerases having desirable properties such as increased polymerase efficiency and speed, increased affinity to DNA substrates and/or increased resistance to DNA polymerase inhibitors. In certain embodiments, the invention provides methods of producing, amplifying and/or sequencing nucleic acid molecules (particularly cDNA molecules) using kits, compositions and/or reactions mixtures containing such novel DNA polymerases.

phi29 DNA polymerase mutants having increased thermostability and processivity

A restriction endonuclease with a recognition sequence 5'-TCGA-3&#x2032;. 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&#x2032;-TCGA-3&#x2032; are also disclosed.

Restriction endonucleases and their uses

The present invention provides a process for the production of nucleic acid encoding a target protein, which comprises: (a) providing an array of RNA or DNA molecules including one or more encoding the target protein; (b) generating a target protein from the array to form RNA-protein or DNA-protein complexes in which the RNA or DNA molecule is non-covalently or covalently bound to the complex; (c) separating the complexes into compartments wherein most or all of the compartments contain no more than one complex; (d) subjecting the complexes to reaction conditions which allow target protein activity; and (e) selecting nucleic acid encoding the target protein on the basis of the activity associated therewith, wherein when the complex is a DNA-protein complex in which the DNA is non-covalently bound, step b) is performed in the absence of separate compartments for each complex.

Growing community of inventors

Vilnius, Lithuania

Remigijus Skirgaila