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Endonuclease and application thereof

A technology of endonuclease and amino acid, applied in the direction of hydrolase, introduction of foreign genetic material by using carrier, recombinant DNA technology, etc., to achieve the effect of broad application prospects

Pending Publication Date: 2022-01-18
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of the above deficiencies in the prior art, the object of the present invention is to provide an endonuclease and its application, aiming at solving the problem that existing enzymes that can specifically recognize deoxyuridine in DNA cannot complete the cleaving of the DNA alone

Method used

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  • Endonuclease and application thereof
  • Endonuclease and application thereof
  • Endonuclease and application thereof

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Embodiment approach

[0045] In one embodiment, the step of using the endonuclease to recognize deoxyuracil in the DNA containing deoxyuracil and cleave the first phosphodiester bond at the 3' end of the deoxyuracil comprises:

[0046] Provide DNA and endonuclease containing deoxyuracil;

[0047] The endonuclease, the DNA containing deoxyuracil, Tris hydrochloride, sodium chloride, dithiothreitol and glycerin are mixed and incubated, and the endonuclease is effective against the The deoxyuracil in the deoxyuracil-containing DNA recognizes and cleaves the first phosphodiester bond at the 3' end of the deoxyuracil.

[0048] The present invention will be further described below by specific examples.

Embodiment 1

[0050] The base sequence of the single-stranded DNA substrate in the present embodiment is shown in Table 1 below:

[0051] Table 1. Base sequences of single-stranded DNA substrates

[0052]

[0053] (1) Synthesis of the endonuclease (the amino acid sequence of which is shown in SEQ ID NO: 1 and 2): the DNA encoding the endonuclease is synthesized through the artificial gene synthesis of the commercial company Beijing Liuhe Huada Gene Technology Co., Ltd. The sequence is inserted into an expression plasmid, and then heterologously expressed in Escherichia coli, and purified by nickel ion affinity chromatography to obtain a pure protein (the endonuclease) for use.

[0054] (2) Synthetic single-stranded DNA substrate:

[0055] Synthesize 58 bases, the 23rd position is deoxyuridine (dU), thymidine (A), deoxyinosine (dI), apurine pyrimidine (AP), and the 5' end has FAM fluorescently labeled single-stranded DNA substrates 1, 2, 3, 4. The base sequences of single-stranded DNA ...

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Abstract

The invention discloses endonuclease and application thereof. An amino acid sequence of the endonuclease is shown as SEQ ID NO: 1 or SEQ ID NO: 2, or the amino acid sequence of the endonuclease and an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 have at least 70% of sequence identity. The endonuclease can be used for directly and specifically recognizing deoxyuracil (deoxyuridine) on a DNA strand and cutting a first phosphodiester bond located at a 3'-terminal of the deoxyuracil is cut, cutting can be independently completed without combined action with other endonucleases with AP site lyase activity, and the endonuclease has wide application prospects in the fields of in-vitro molecular diagnosis of DNA damage mutation and the like. In addition, the invention provides a cutting site, i.e., the first phosphodiester bond located at the 3'-terminal of the deoxyuracil for the first time, and the cutting site is not reported in the prior art.

Description

technical field [0001] The invention relates to the field of endonucleases, in particular to an endonuclease and its application. Background technique [0002] Endonuclease (endonuclease) is an enzyme that can hydrolyze the internal phosphodiester bonds of nucleic acid molecular chains to generate oligonucleotides. From the perspective of substrate specificity, it can be divided into enzymes that decompose DNA such as DNaseI and DNaseII; enzymes that decompose RNA such as RNase and RNaseT1. Most general endonucleases are not base-specific, and a special case is restriction endonucleases, which can recognize and cut specific base sequences. Nucleases involved in DNA damage repair in living organisms can also specifically recognize erroneous bases, hydrolyze erroneous bases or cut nucleic acid strands containing erroneous bases, thereby causing a series of downstream enzyme reactions and further Correct mistakes. The nucleases capable of specifically recognizing damaged bas...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/70
CPCC12N9/22C12N15/70
Inventor 李猛李锦权
Owner SHENZHEN UNIV
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