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MODIFIED TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) ENZYMES

A deoxynucleotide and transferase technology, applied in the field of application, can solve problems such as the effective addition of TdT that has not yet been shown

Pending Publication Date: 2021-09-28
NUCLERA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, TdT has not been shown to efficiently add nucleoside triphosphates containing a 3′-O-reversible termination moiety to build nascent single-stranded DNA strands necessary for the de novo synthesis cycle

Method used

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  • MODIFIED TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) ENZYMES
  • MODIFIED TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) ENZYMES
  • MODIFIED TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) ENZYMES

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0300] Expression of TdT variants

[0301] Briefly, a plasmid containing the gene encoding terminal transferase was transformed into BL21 E. coli competent cells. Starting Luria Broth (LB) cultures were grown overnight at 37°C and inoculated into LB expression cultures. Expression cultures were grown to an optical density at 600 nm of 0.6 and induced by adding IPTG to 1 mM. Cultures were induced and grown overnight at 25°C. The next morning, cultures were lysed in detergent lysis buffer and purified to homogeneity by immobilized metal affinity chromatography (IMAC).

[0302] Determination of incorporation of reversible terminators by TdT variants

[0303] 173 terminal transferases were expressed, purified and compared to wild type bovine TdT (SEQ ID NO 2). The purified engineered TdT was then used in the following assay: fluorescently labeled 15-nt ssDNA primers were mixed with 1×TdT buffer (Thermo Fisher Scientific), yeast inorganic pyrophosphatase (Sigma-Aldrich, 0.1 mU / μl...

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Abstract

The invention relates to engineered terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of Polmu, Polbeta, Pollambda, and Poltheta of any species or the homologous amino acid sequence of X family polymerases of any speciesand their use in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids,and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. Theinvention also relates to the use of new terminal deoxynucleotidyl transferases and 3'-blockednucleoside triphosphates in a method of template independent nucleic acid synthesis.

Description

[0001] field of invention [0002] The present invention relates to specific terminal deoxynucleotidyl transferase (TdT) or the homologous amino acid sequence of Polμ, Polβ, Polλ and Polθ of any species or the homologous amino acid sequence of X family polymerase of any species in the method for nucleic acid synthesis Uses relate to a method for synthesizing nucleic acid, and relate to the use of a kit comprising said enzyme in a method for nucleic acid synthesis. The present invention also relates to the use of terminal deoxynucleotidyl transferase or homologous enzymes and 3'-blocked nucleoside triphosphates in a method for template-independent nucleic acid synthesis. [0003] Background of the invention [0004] Nucleic acid synthesis is essential to modern biotechnology. The ability of the scientific community to artificially synthesize DNA, RNA and proteins has enabled the rapid development of the field of biotechnology. [0005] Artificial DNA synthesis allows biotech a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12
CPCC12N9/1264C12Y207/07031
Inventor 迈克尔·春·浩·陈戈登·罗斯·米钦罗伊伊恩·哈斯顿·库克陈思红
Owner NUCLERA LTD
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