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 - 58 . (canceled)
59 . A polymerase comprising the amino acid sequence Ile-Gly-Asp-Arg-Ala-Ile-Pro at the positions functionally equivalent to residues 710-716 of SEQ ID NO:22, having a substitution mutation at the position functionally equivalent to Arg713 to a nonpolar amino acid, whereby the polymerase has a reduced affinity for DNA.
60 . The polymerase of claim 59 , wherein the polymerase is capable of incorporating a nucleotide or nucleotides into a plurality of separate DNA templates in each reaction cycle as compared to a control polymerase, wherein the control polymerase is the unaltered polymerase and is capable of incorporating a nucleotide or nucleotides into a single DNA template in each reaction cycle.
61 . The polymerase of claim 59 , wherein the polymerase is capable of forming an increased number of productive polymerase-DNA complexes in each reaction cycle as compared to a control polymerase, wherein the control polymerase is the unaltered polymerase.
62 . The polymerase of claim 59 , wherein the affinity of the polymerase for nucleotides and the fidelity of the polymerase is substantially unaffected by the substitution mutation.
63 . The polymerase according to claim 59 , wherein the substitution mutation converts the position functionally equivalent to Arg713 to glycine (G) or methionine (M).
64 . The polymerase according to claim 59 , wherein the substitution mutation converts the position functionally equivalent to Arg713 to alanine (A).
65 . A kit for performing a nucleotide incorporation reaction comprising: a polymerase as defined in claim 59 , 63 , or 64 , and a nucleotide solution.
66 . The kit of claim 65 , wherein the nucleotide solution comprises labelled nucleotides.
67 . The kit of claim 65 , wherein the nucleotides comprise synthetic nucleotides.
68 . The kit of claim 65 , wherein the nucleotides comprise modified nucleotides.
69 . The kit of claim 68 , 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.
70 . The kit according to claim 69 , wherein the modified nucleotides are 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 , preferably OH, 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.
71 . The kit according to claim 70 , wherein R′ of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.
72 . The kit according to claim 71 , wherein —Z of the modified nucleotide or nucleoside is of formula —C(R′).sub.2-N.sub.3.
73 . The kit according to claim 72 , wherein Z is an azidomethyl group.
74 . The kit according to claim 69 , wherein the modified nucleotides are fluorescently labelled to allow their detection.
75 . The kit according to claim 69 , 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 comprising 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.
76 . The kit according to claim 75 , wherein the detectable label comprises a fluorescent label.
77 . The kit of claim 65 further comprising one or more DNA template molecules and/or primers.