Enzyme composition for dna end repair, adenylation, phosphorylation

An enzyme composition and end repair technology, applied in the field of enzyme compositions, can solve problems such as damage to genome sequence assembly, complex genome sequence assembly, damage to genome assembly and the like

Active Publication Date: 2020-04-10
THERMO FISHER SCI LITHUANIA HLDG UAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the library preparation contains many such chimeric molecules, they may subsequently complicate or impair the assembly of the genome sequence
Chimeric DNA molecules may impair assembly of sequenced genomes in NGS applications

Method used

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  • Enzyme composition for dna end repair, adenylation, phosphorylation
  • Enzyme composition for dna end repair, adenylation, phosphorylation
  • Enzyme composition for dna end repair, adenylation, phosphorylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Prove that different enzymes require different buffers

[0044] It is known in the art that each enzyme has different optimal storage and reaction conditions. Enzyme storage conditions are often different from the recommended reaction conditions shown in Table 2. Table 2 shows the storage and reaction buffer components of DNA inactivation, phosphorylation, and dA tailing enzymes sold by the following commercial suppliers: Thermo Fisher Scientific, New England Biolabs and Life Technologies.

[0045] Table 2. Storage buffer (SB) and reaction buffer (RB) of enzymes used for passivation, phosphorylation and dA tailing of DNA fragments

[0046]

[0047]

[0048]

[0049] In order to obtain the composition of the present invention, all the above-indicated enzymes T4 DNA polymerase, Klenow fragment, T4 DNA polynucleotide kinase and modified thermophilic polymerases with end-tailing activity, such as modified Brucella The thermobacterium or Thermus aquaticus DNA polymerase must be p...

Embodiment 2

[0054] Effect of buffer and polymerase composition on passivation, tailing and phosphorylation

[0055] To test the efficiency of 3'end tailing of DNA fragments and analyze the potential bias of the 3'end nucleotides, we developed and used a kind of different length and 3'end nucleotides, and used it at the 5'end Cy5 labeled four oligonucleotide duplex model system ( figure 1 ).

[0056] Check Klenow fragment exo - The mutant’s dA tailing ability and the figure 2 It is shown that lane (A) is the best Klenow fragment buffer (10x reaction buffer, EP0421) supplemented with 0.2mM dATP; lane (B) is commercial buffer G containing 1mM DTT and 0.2mM dATP; and lane ( C) is the optimized DNA end repair buffer (fast DNA end repair kit K0771). Use 5 units of enzyme in a 50μl reaction mixture (containing as figure 1 A dA tailing reaction was performed in the shown Cy-5 labeled oligonucleotide duplex (7.5 pmol equivalent molecular mixture). The pair of bands represents the oligonucleotide d...

Embodiment 3

[0071] Stability of 2x concentrated terminal transformation master mix

[0072] For stability testing, the 2x terminal transformation master mix as disclosed was stored at -20°C and +25°C (to represent accelerated stability testing) for different periods of time. use for Figure 5 Perform stability testing with the same experimental protocol shown in. Incubate 1 μg control DNA fragment in 50 μl reaction mixture of 1x end transformation master mix that has been stored at -20°C for 5 min to allow end repair enzymes to inactivate and phosphorylate the DNA ends, and then incubate at 72°C for 10 minutes. Conducive to the simultaneous inactivation of the mesophilic DNA end repair enzyme and the 3'-end tailing of the DNA by mod-Tbr DNA polymerase. After this step, a DNA adaptor with a length of 60 bp carrying a 3'terminal dT extension was added to the reaction mixture to a final concentration of 1 μM, and ligation was performed at 22° C. for 5 min using T4 DNA ligase. The resulting re...

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Abstract

Enzyme compositions and their method of use that provide ready-to-use master mixtures. The compositions comprise a modified thermophilic DNA polymerase lacking 5′-3′ and 3′-5′ exonuclease activity premixed with T4 DNA polymerase, Klenow fragment and T4 polynucleotide kinase and all other necessary components, including reaction buffer and nucleoside triphosphates, required to perform DNA blunting, phosphorylation, and single nucleotide extension reactions in one tube and in two steps. Among other benefits, the mixture of different enzymes, buffers and nucleoside triphosphates is stable during prolonged storage.

Description

[0001] This application claims priority to the U.S. Patent Application Serial No. 61 / 882,480 filed on September 25, 2013 and the U.S. Patent Application Serial No. 61 / 904,543 filed on November 15, 2013. Each patent application is incorporated herein by reference in its entirety. . Technical field [0002] The present invention relates to enzyme compositions, and more specifically to enzyme compositions that provide ready-to-use master mixes and methods of use thereof. Background technique [0003] Several methods for high-throughput DNA sequencing (Nature.437,376-380(2005); Science.309(2005)728-1732) rely on universal amplification reactions, whereby DNA samples are randomly divided into fragments and then processed , So that the ends of different fragments all contain the same DNA sequence. Fragments with universal ends can be amplified in a single reaction with a single pair of amplification primers. Separating the fragment library into single molecule levels before amplificat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12Q1/6869C12N15/10C40B40/06
CPCC12N9/1205C12N9/1241C12N9/1252C12N9/22C12N9/96C12Y207/01078C12N11/18
Inventor J.卢比恩A.贝雷斯尼亚科瓦斯A.卢比斯
Owner THERMO FISHER SCI LITHUANIA HLDG UAB
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