Modified polymerases for improved incorporation of nucleotide analogues
The invention relates to modified polymerase enzymes which exhibit improved incorporation of nucleotide analogues bearing substituents at the 3′ position of the sugar moiety that are larger in size than the naturally occurring 3′ hydroxyl group. Also described are methods of using the polymerases to incorporate nucleotides into polynucleotides, particularly in the context of DNA sequencing.
1. An altered Pfu or JDF-3 DNA polymerase, wherein the polymerase comprises two substitution mutations at the first and second, the first and third, or the second and third amino acids of the motif A region, wherein the first amino acid of the motif A region is mutated to tyrosine (Y), phenylalanine (F), isoleucine (I), alanine (A), valine (V), glutamine (Q), cysteine (C), or serine (S), the second amino acid of the motif A region is mutated to alanine (A), serine (S), or glycine (G), and the third amino acid of the motif A region is mutated to serine (S), alanine (A), glycine (G), or an amino acid having a beta-branched side chain, and wherein the polymerase is capable of incorporating a 3′ O-azidomethyl-modified nucleotide.
2. The polymerase of claim 1 , wherein the two substitution mutations are at the first and second amino acids of the motif A region, wherein the first amino acid of the motif A region is mutated to tyrosine (Y), phenylalanine (F), or valine (V), and the second amino acid of the motif A region is mutated to alanine (A).
3. An altered Pfu or JDF-3 DNA polymerase, wherein the polymerase comprises three substitution mutations at the motif A region, wherein the first amino acid of the motif A region is mutated to tyrosine (Y), phenylalanine (F), isoleucine (I), alanine (A), valine (V), glutamine (Q), cysteine (C), or serine (S), the second amino acid of the motif A region is mutated to alanine (A), serine (S), or glycine (G), and the third amino acid of the motif A region is mutated to serine (S), alanine (A), glycine (G), or an amino acid having a beta-branched side chain, and wherein the polymerase is capable of incorporating a 3′ O-azidomethyl- modified nucleotide.
4. The polymerase of claim 3 , wherein the three substitution mutations of the motif A region are YST, FAI, AAA, YAS, YAV, YGI, YSG, SGG, CST, IAL, CGG, SAL, SAA, CAA, YAA, QAS, VSS, VAG, VAV, FAV, AGI, YSS, AAT, FSS, or VAL.
5. The polymerase of claim 3 , wherein the three substitution mutations of the motif A region are FAP, VAP, YST, FAI, AAA, YAS, YAV, YGI, YSG, SGG, CST, IAL, CGG, SAL, SAA, CAA, YAA, QAS, VSS, VAG, VAV, FAV, AGI, YSS, AAT, FSS or VAL, and the polymerase comprises a substitution mutation at the second amino acid at the motif B region, wherein the substitution mutation is to leucine (L).
6. The polymerase of claim 3 , wherein the polymerase lacks 3′-5′ exonuclease activity.
7. The polymerase according to claim 3 , further comprising at least one amino acid substitution mutation in the motif B region, whereby the activity of the polymerase is enhanced.
8. The polymerase according to claim 7 , wherein the substitution mutation in the motif B region is to the second amino acid.
9. The polymerase according to claim 8 , wherein the substitution mutation is to an amino acid selected from the group consisting of F, I, S, V and C.
10. The polymerase according to claim 8 , wherein the substitution mutation is to L.
11. The polymerase according to claim 7 , wherein the motif B substitution mutations are to G at the first amino acid position and L at the third.
12. The polymerase according to claim 7 , wherein the motif B substitution mutations are to N at the first amino acid position and Q at the third.
13. The polymerase according to claim 7 , wherein the motif B substitution mutations are to those substitutions selected from the group consisting of SKN, GRD, KHN, ISN and THH.
14. The polymerase of claim 3 , wherein the three substitution mutations of the motif A region are AAI.
15. The polymerase of claim 3 , wherein the three substitution mutations of the motif A region are AAI, and the polymerase comprises a substitution mutation at the second amino acid at the motif B region, wherein the substitution mutation is to valine (V).
16. The polymerase of claim 1 , wherein the amino acid having a beta-branched side chain is isoleucine (I), threonine (T), valine (V), or leucine (L).
17. The polymerase of claim 3 , wherein the amino acid having a beta-branched side chain is isoleucine (I), threonine (T), valine (V), or leucine (L).
18. The polymerase of claim 2 , wherein the polymerase lacks 3′-5′ exonuclease activity.