Polymerase

Abstract

The present invention relates to an engineered polymerase with an expanded substrate range characterised in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase as compared with the wild type polymerase from which it is derived.

1 . An engineered polymerase with an expanded substrate range characterised in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase as compared with the wild type polymerase from which it is derived. 
     
     
         2 . The engineered polymerase according to  claim 1  wherein the agent-labelled nucleotide analogue is a dye labelled nucleotide analogue. 
     
     
         3 . The engineered polymerase according to  claim 2  wherein the agent-labelled nucleotide analogue is a fluorescent labelled nucleotide analogue. 
     
     
         4 . The engineered polymerase according to  claim 3  wherein the fluorescent label is selected from the group consisting of Alexa Fluor™ dye, diethylaminocoumarin, tetramethylrhodamine, N-methylanthraniloyl, trinitrophenyl, etheno derivatives, biotin, fluorescein, or carbocyanine. 
     
     
         5 . The engineered polymerase according to  claim 4  wherein the agent-labelled nucleotide analogue is a carbocyanine labelled nucleotide analogue. 
     
     
         6 . The engineered polymerase according to  claim 1  which is a DNA polymerase. 
     
     
         7 . The engineered polymerase according to  claim 2 , wherein the dye labelled nucleotide analogue is selected from the group consisting of Cy5-dNTP and Cy3-dNTP. 
     
     
         8 . (canceled) 
     
     
         9 . The engineered polymerase according to  claim 6  wherein the polymerase has one or more of the following mutations: V337I, E399D, N400D, R407I and/or Y546H. 
     
     
         10 . The engineered polymerase according to  claim 1  comprising the amino acid sequence designated SEQ ID NO: 1. 
     
     
         11 . The engineered polymerase according to  claim 1  comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 4, 6, 8, 10, 12 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40. 
     
     
         12 . The isolated nucleic acid molecule according to  claim 17  comprising a nucleotide sequence encoding an engineered polymerase and selected from the group consisting of SEQ ID NOS: 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39. 
     
     
         13 . The engineered DNA polymerase according to  claim 6  wherein the engineered DNA polymerase is derived from wild type DNA polymerase by substitution, deletion or insertion of one or more amino acids. 
     
     
         14 . The engineered DNA polymerase according to  claim 6 , wherein the engineered DNA polymerase is derived from a DNA polymerase by substitution, deletion or insertion of one or more amino acids. 
     
     
         15 . The engineered polymerase according to  claim 14 , wherein the DNA polymerase is selected from the group consisting of polA polymerases, polB polymerases and pfu polymerases. 
     
     
         16 . The engineered polymerase according to  claim 1 , wherein the amino acid sequence of the polymerase is at least 70% identical to that designated herein as E1O and set forth in SEQ ID NO: 1. 
     
     
         17 . An isolated nucleic acid molecule which encodes an engineered polymerase polypeptide wherein the nucleotide, sequence is at least 80% identical to any of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39, and wherein said polypeptide is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase. 
     
     
         18 .- 21 . (canceled) 
     
     
         22 . A method for the incorporation of detection agent-labelled nucleotide analogues into newly synthesised nucleic acid which method comprises the use of an engineered polymerase according to  claim 1 . 
     
     
         23 . (canceled) 
     
     
         24 . The method according to  claim 22  wherein the detection agent-labelled nucleotide is a dye-labelled nucleotide analogue. 
     
     
         25 . The method according to  claim 24  wherein the dye-labelled nucleotide analogue is a fluorescent dye-labelled nucleotide analogue. 
     
     
         26 . The method according to  claim 25  wherein the fluorescent dye-labelled nucleotide analogue is selected from the group consisting of the following: Cy3-CTP, Cy5-CTP. 
     
     
         27 .- 38 . (canceled)