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Improved polymerases

A polymerase and amino acid technology, applied in the field of polymerase, can solve research problems

Active Publication Date: 2008-05-14
ILLUMINA CAMBRIDGE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Thumb domains from thermophilic archaeon polymerases have not been studied before

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0195] Example 1 - Preparation of altered polymerases

[0196] principle

[0197] A site-directed mutation was introduced in the C-terminal region of the 9°N-7YAV C223S polymerase, thereby reducing the affinity of the enzyme to DNA (wild-type 9°N-7 polymerase has a very high DNA affinity, Kd=50pM; Southworth et al. 1996 .PNAS.93, 5281).

[0198]An energy-minimized overlap alignment using the crystal structure of the open form of the 9°N-7 DNA polymerase (PDB=1qht), the open structure of the closely related DNA polymerase RB69 (PDB=1ih7), and the closed form of RB69 (PDB=1ig9) ( by Cresset) as a structural model to identify key residues involved in DNA binding. The crystal structure of the occluded form of RB69 polymerase (Franklin et al. 2001. Cell 105, 657) identified a number of residues that form hydrogen bonds or electrostatic interactions with complexed DNA, either directly with the nucleotide bases or with the phosphate backbone. A significant number of these resi...

Embodiment 2

[0222] Example 2 - NUNC tube assay using crude protein preparation.

[0223] A 5 ml small culture of the mutant enzyme (while using a YAV C223S exo-culture for direct comparison) was subjected to flash purification up to the heat treatment step as described in WO2005 / 024010. At this point, the sample was considered pure enough to be tested for activity.

[0224] Each crude preparation was buffer exchanged with enzymatic buffer (50 mM Tris pH 8.0, 6 mM MgSO4, 1 mM EDTA, 0.05% Tween20) using a S300 gel filtration spin column. This sample was not normalized to concentration. The assay used was simple incorporation of ffTTP into surface coupled A template hairpins. According to the product instructions, 2 pmol of 5'-amino oligomer 815

[0225] (5'-CGATCACGATCACGATCACGATCACGATCACGATCACGATCACGCTGATGTGCATGCTGTTGTTTTTTTACAACAGCATGCACATCAGCG-3') (SEQ ID NO: 12)

[0226] Coupled with NUNC-nucleolink gel strips.

[0227] After washing, each well was incubated with 20 μl aliquots o...

Embodiment 3

[0241] Example 3 - Single base incorporation assay

[0242] The activity of crude enzyme preparations (normalized concentrations) was measured using the single base incorporation assay described in WO2005 / 024010. In 2μM ffT-N3-cy3 and 20nM 10A hairpin DNA ( 32 P-labeled) were incubated with 30 or 3 μg / ml crude enzyme preparation for 10 min, and aliquots of the reaction mixture were removed at 0, 30, 60, 180 and 600 sec and run on a 12% acrylamide gel. Electrophoresis.

[0243] result

[0244] The gel image is shown in Figure 3.

[0245] Use Imagequant to quantify the band intensity, and the fluorescence intensity plotted against the incubation time is shown in Figure 4 time course.

[0246] These data give an estimate of the ffTTP first base incorporation performance of the mutant enzymes relative to YAV. Due to normalization to concentration, the activities are directly comparable. The Δ71 mutant was essentially inactive (kobs 21% of that observed with YAV), R743A a...

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Abstract

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.

Description

technical field [0001] The present invention relates to polymerases, and more particularly to modified DNA polymerases having an affinity for DNA such that the polymerases are able to incorporate nucleotides into multiple separate DNA templates per reaction cycle, And can form an increased number of productive polymerase-DNA complexes in each reaction cycle. Also within the scope of the present invention are methods of using said modified polymerase for sequencing, especially in clustered arrays. Background technique [0002] The three-dimensional crystal structures of certain DNA polymerases are shown as three separate subdomains called palm, finger, and thumb (Joyce, C.M. and Steiz, T.A. (1994) Function and structure relationships in DNA polymerases, Annu. Rev. Biochem., 63, 777-822), each subdomain plays a key role during DNA polymerization. [0003] The C-terminal thumb domain of DNA polymerases has been found to be associated with DNA binding and processivity (Doublie...

Claims

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Application Information

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IPC IPC(8): C12N9/12
Inventor 托拜厄斯·威廉·巴尔·奥斯特杰弗里·保罗·史密斯尚卡尔·巴拉苏布拉马尼亚姆罗伯托·里加蒂拉克尔·马里亚·桑切斯
Owner ILLUMINA CAMBRIDGE LTD
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