Polymerases
Modified DNA polymerases have an affinity for DNA such that the polymerase has an ability to incorporate one or more nucleotides into a plurality of separate DNA templates in each reaction cycle. The polymerases are capable of forming an increased number of productive polymerase-DNA complexes in each reaction cycle. The modified polymerases may be used in a number of DNA sequencing applications, especially in the context of clustered arrays.
1. An altered family B polymerase having a reduced affinity for DNA, wherein the polymerase comprises at least two substitution mutations selected from the positions functionally equivalent to Lys705, Arg713, and Arg743 of the 9° N DNA polymerase amino acid sequence of SEQ ID NO:22.
2. The altered polymerase of claim 1 , wherein the polymerase comprises substitution mutations at the positions functionally equivalent to Lys705, Arg713, and Arg743 of the 9° N DNA polymerase amino acid sequence of SEQ ID NO:22.
3. The altered polymerase of claim 2 , wherein each substitution mutation is to a non-polar amino acid.
4. The altered polymerase of claim 3 , wherein the non-polar amino acid is alanine, glycine, or methionine.
5. The altered polymerase of claim 1 , wherein the polymerase further comprises a substitution mutation at the position functionally equivalent to Arg606, His679, or the combination thereof, of the 9° N DNA polymerase amino acid sequence of SEQ ID NO:22.
6. The altered polymerase of claim 1 , wherein the polymerase further comprises a substitution mutation at the position functionally equivalent to Leu408, Tyr409, Pro410, or a combination thereof, of the 9° N DNA polymerase amino acid sequence of SEQ ID NO:22.
7. The altered polymerase of claim 1 , wherein the polymerase is a family B archaeal polymerase.
8. A kit for performing a nucleotide incorporation reaction, said kit comprising: a polymerase as defined in claim 1 and a nucleotide solution.
9. The kit of claim 8 , wherein the nucleotide solution comprises labelled nucleotides.
10. The kit of claim 8 , wherein the nucleotides comprise synthetic nucleotides.
11. The kit of claim 8 , wherein the nucleotides comprise modified nucleotides.
12. The kit of claim 11 , wherein the modified nucleotides have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group.
13. The kit of claim 11 , wherein modified nucleotides comprise a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure
—O—Z
wherein Z is any of —C(R′) 2 —O—R″, —C(R′) 2 —N(R″) 2 , —C(R′) 2 —N(H)R″, C(R′) 2 —S—R″, and —C(R′) 2 —F,
wherein each R″ is or is part of a removable protecting group;
each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido group, or a detectable label attached through a linking group; or (R′) 2 represents an alkylidene group of formula ═C(R′″) 2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and
wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′) 2 —F, the F is exchanged for OH, SH or NH 2 , which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH;
with the proviso that where Z is —C(R′) 2 —S—R″, both R′ groups are not H.
14. The kit of claim 13 , wherein R′ of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.
15. The kit of claim 14 , wherein Z is of formula —C(R′) 2 —N 3 .
16. The kit of claim 14 , wherein Z is an azidomethyl group.
17. The kit of claim 11 , wherein the modified nucleotides are fluorescently labelled to allow their detection.
18. The kit of claim 11 , wherein the modified nucleotides comprise a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, wherein the cleavable linker contains a moiety selected from the group consisting of:
wherein X is selected from the group consisting of O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside.
19. The kit of claim 18 , wherein the detectable label comprises a fluorescent label.
20. The kit of claim 8 , further comprising one or more DNA template molecules, primers, or a combination thereof.