Phage Phi 29 DNA Polymerase Chimera

Abstract

A DNA polymerase chimera comprising an amino-terminal (N-terminal) region encoding a φ29 type DNA polymerase and a carboxyl-terminal (C-terminal) region comprising at least one HhH domain which are bound by a connecting amino acid sequence is disclosed along with and the use thereof for replicating, amplifying or sequencing a template DNA. Also disclosed is a method for replicating, amplifying or sequencing a deoxyribonucleic acid with a DNA polymerase chimera and kits for carrying out the methods.

1 . A DNA polymerase chimera comprising:
 a) an amino acid sequence encoding a phi29 (φ29) type DNA polymerase, bound by its C-terminal end to   b) a connecting amino acid sequence bound by its C-terminal end to   c) an amino acid sequence comprising at least one helix-hairpin-helix (HhH) domain.   
     
     
         2 . The DNA polymerase chimera according to  claim 1 , wherein the amino acid sequence of (c) comprises at least one HhH domain of a protein which is selected from the list comprising:
 topoisomerase V of  Methanopyrus kandleri,      MutY, Nth, MutM/Fpg, Nei, UvrC, DinP, RecR, UmuC, DnaE or DnIJ of  Escherichia coli,      RAD1, RAD2, RAD10, RAD27, RAD 55, RAD 57, REV1, OGG1, NTG1, NTG2, DIN-7 or EXO-1 of yeasts, or   a homologous protein of the above in  Bacillus subtilis, Caenorhabditis elegans, Haemophilus influenzae, Methanococcus jannaschii, Micrococcus luteus, Methanobacterium thermoformicum  or  Salmonella typhimurium.      
     
     
         3 . The DNA polymerase chimera according to  claim 2 , wherein the amino acid sequence of (c) comprises at least one HhH domain derived from the topoisomerase V of  Methanopyrus kandleri.    
     
     
         4 . The DNA polymerase chimera according to  claim 3 , wherein the amino acid sequence of (c) is SEQ ID NO: 3. 
     
     
         5 . The DNA polymerase chimera according to  claim 3 , wherein the amino acid sequence of (c) is SEQ ID NO: 3 bound by its C-terminal end to SEQ ID NO: 4. 
     
     
         6 . The DNA polymerase chimera according to  claim 1 , wherein the connecting amino acid sequence of (b) is SEQ ID NO: 5 or SEQ ID NO: 6. 
     
     
         7 . The DNA polymerase chimera according to  claim 1 , wherein the φ29 type DNA polymerase of (a) is selected from the group of DNA polymerases isolated from the following phages: φ29, Cp-1, PRD-1, φ15, φ21, PZE, PZA, Nf, M2Y, B103, GA-1, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17 and ABV. 
     
     
         8 . The DNA polymerase chimera according to  claim 1 , wherein the φ29 type DNA polymerase of (a) has an amino acid sequence having an identity of at least 80% with SEQ ID NO: 1. 
     
     
         9 . The DNA polymerase chimera according to  claim 8 , wherein the φ29 type DNA polymerase of (a) has an amino acid sequence having an identity of at least 90% with SEQ ID NO: 1. 
     
     
         10 . The DNA polymerase chimera according to  claim 9 , wherein the φ29 type DNA polymerase of (a) has the amino acid sequence SEQ ID NO: 1. 
     
     
         11 . The DNA polymerase chimera according to  claim 1 , wherein the φ29 type DNA polymerase of (a) has a modification in the exonuclease domain and wherein said modified DNA polymerase has less than 10% of exonuclease activity than the corresponding naturally occurring DNA polymerase. 
     
     
         12 . The DNA polymerase chimera according to  claim 11 , wherein the modified φ29 type DNA polymerase of (a) has less than 1% of exonuclease activity than the corresponding naturally occurring DNA polymerase. 
     
     
         13 . The DNA polymerase chimera according to  claim 12 , wherein the modified φ29 type DNA polymerase of (a) lacks detectable exonuclease activity with respect to the corresponding naturally occurring DNA polymerase. 
     
     
         14 .- 51 . (canceled) 
     
     
         52 . A kit for replicating, amplifying or sequencing a template DNA comprising:
 a) a DNA polymerase chimera comprising
 (i) an amino acid sequence encoding a phi29 (φ29) type DNA polymerase, bound by its C-terminal end to 
 (ii) a connecting amino acid sequence bound by its C-terminal end to 
 (iii) an amino acid sequence comprising at least one helix-hairpin-helix (HhH) domain 
   b) a buffer, and   c) magnesium chloride.   
     
     
         53 . The kit according to  claim 52 , further comprising polyoxyethylenated sorbitan monolaurate. 
     
     
         54 . The kit according to  claim 52 , further comprising an ammonium salt. 
     
     
         55 . The kit according to  claim 52 , further comprising a potassium salt. 
     
     
         56 . The kit according to  claim 52 , further comprising a primer. 
     
     
         57 . The kit according to  claim 56 , wherein the primer is arbitrary and is protected against the action of exonucleases. 
     
     
         58 . The kit according to  claim 52 , further comprising nucleoside triphosphates. 
     
     
         59 . The kit according to  claim 56 , wherein at least one nucleoside triphosphate or one primer is labelled.