Dna polymerases with improved activity

Abstract

Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

1 .- 17 . (canceled) 
     
     
         18 . A DNA polymerase having increased reverse transcriptase efficiency, compared with a control DNA polymerase, wherein the DNA polymerase has at least 90% amino acid sequence identity to SEQ ID NO:6 and wherein the amino acid of the DNA polymerase corresponding to position 522 of SEQ ID NO:6 is any amino acid other than E, and wherein the control DNA polymerase has the same amino acid sequence as the DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 522 of SEQ ID NO:6 is E. 
     
     
         19 . The DNA polymerase of  claim 18 , wherein the amino acid of the DNA polymerase corresponding to position 522 of SEQ ID NO:6 is selected from G, A, L, M, F, W, P, S, T, C, Y, Q, D, K, R, V, I, N, or H. 
     
     
         20 . The DNA polymerase of  claim 18 , comprising a motif in the polymerase domain comprising
 S-T-X 1 -X 2 -X 3 -X 4 -L-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -H-X 11 -I, wherein:   X 1  is S, R, N or A;   X 2  is A, I or V;   X 3  is A, E, S or D;   X 4  is V or A;   X 5  is any amino acid other than E;   X 6  is A, L, E, P or K;   X 7  is L or I;   X 8  is R, A or S;   X 9  is E, G, N, K, D or P;   X 10  is A, E, H or Y;   X 11  is P or E (SEQ ID NO:8).   
     
     
         21 . The DNA polymerase of  claim 20 , wherein X 5  is selected from G, A, L, M, F, W, P, S, T, C, Y, Q, D, K, R, V, I, N, or H. 
     
     
         22 . The DNA polymerase of  claim 18 , comprising a motif in the polymerase domain comprising
 S-T-X 1 -X 2 -X 3 -V-L-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -H-X 11 -I, wherein:   X 1  is S or R;   X 2  is A or I;   X 3  is A or E;   X 5  is any amino acid other than E;   X 6  is A, L or E;   X 7  is L or I;   X 8  is R or A;   X 9  is E, G or N;   X 10  is A or E;   X 11  is P or E. (SEQ ID NO:9).   
     
     
         23 . The DNA polymerase of  claim 18 , comprising a motif in the polymerase domain comprising
 S-T-S-A-A-V-L-X 5 -A-L-R-E-A-H-P-I, wherein:   X 5  is any amino acid other than E (SEQ ID NO:10).   
     
     
         24 . The DNA polymerase of  claim 23 , comprising a motif in the polymerase domain comprising
 S-T-S-A-A-V-L-X 5 -A-L-R-E-A-H-P-I, wherein:   X 5  is G (SEQ ID NO:11).   
     
     
         25 . The DNA polymerase of  claim 18 , wherein the amino acid corresponding to position 580 of SEQ ID NO:6 is any amino acid other than D or E. 
     
     
         26 . The DNA polymerase of  claim 18 , wherein the amino acid corresponding to position 580 of SEQ ID NO:6 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K. 
     
     
         27 . The DNA polymerase of  claim 20 , wherein the polymerase has at least 95% amino acid sequence identity to SEQ ID NO:6. 
     
     
         28 . A recombinant nucleic acid encoding the DNA polymerase according to  claim 18 . 
     
     
         29 . A method for conducting primer extension, comprising:
 contacting a DNA polymerase according to claim  1  with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for extension of the primer, thereby producing an extended primer.   
     
     
         30 . The method of  claim 31 , wherein the template is RNA. 
     
     
         31 . The method of  claim 31 , wherein the primer extension method comprises a polymerase chain reaction (PCR). 
     
     
         32 . A kit for producing an extended primer, comprising:
 at least one container providing a DNA polymerase according to  claim 18 .   
     
     
         33 . The kit according to  claim 34 , further comprising one or more additional containers selected from the group consisting of:
 (a) a container providing a primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template;   (b) a container providing nucleoside triphosphates; and   (c) a container providing a buffer suitable for primer extension.   
     
     
         34 . A reaction mixture comprising a DNA polymerase according to  claim 18 , at least one primer, a polynucleotide template, and nucleoside triphosphates. 
     
     
         35 . The reaction mixture of  claim 36 , wherein the polynucleotide template is RNA. 
     
     
         36 . The DNA polymerase of  claim 18 , wherein the polymerase has at least 95% amino acid sequence identity to SEQ ID NO:6. 
     
     
         37 . An expression vector comprising the recombinant nucleic acid of  claim 28 . 
     
     
         38 . A host cell transformed with the expression vector of  claim 37 . 
     
     
         39 . A method for producing a DNA polymerase having increased reverse transcriptase efficiency compared with a control DNA polymerase, the method comprising culturing the host cell of  claim 38  under conditions suitable for expression of the recombinant nucleic acid.