Enzymatic synthesis method for DNA phosphorothioation

A technology of phosphorothiolation and enzymatic synthesis, which is applied in the field of phosphorothiolation modification of DNA molecules, can solve the problem of no DNA phosphorothiolation modification, etc., and achieve the effect of increasing the frequency of modification

Inactive Publication Date: 2014-12-10
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, there is no report of using enzymatic methods to modify DNA by phosphorothioation

Method used

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  • Enzymatic synthesis method for DNA phosphorothioation
  • Enzymatic synthesis method for DNA phosphorothioation
  • Enzymatic synthesis method for DNA phosphorothioation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, whole cell lysate method synthesis phosphorothioylated DNA

[0034] (1), preparation of whole cell lysate

[0035] Inoculated with Salmonella ( Salmonella enterica ) serovar Cerro 87 to 200mL LB medium (Luria-Bertani medium), cultured at 37°C until A 600 is 1.5;

[0036] 4°C, 5000g centrifugation for 10 minutes to collect the bacterial cells, washed with phosphate buffer three times, washed with 20mL reaction buffer (20mM Tris-HCl, pH8.0, 60mM KCl, 10mM MgCl2, 1mM EDTA, 2mM DTT, 1mM PMSF and 25% (V / V) glycerol resuspended for later use;

[0037] The cell resuspension was repeatedly frozen and thawed 3 times, and then ultrasonically disrupted, centrifuged at 15,000 g for 20 minutes at 4°C, and the supernatant was taken. The supernatant is the whole cell lysate used to catalyze the modification of phosphorothioation of DNA.

[0038] (2), preparation of short-chain DNA substrate

[0039] The short-chain DNA synthesized by chemical method is used as the ...

Embodiment 2

[0056] Example 2. Heterologous expression and purification of protein system catalyzed synthesis of phosphorothioylated DNA

[0057] (1) Heterologous expression and purification of protein systems

[0058] from Salmonella ( Salmonella enterica ) serovar Cerro 87 amplifies four genes responsible for DNA phosphorothioylation modification, namely iscS, dptC, dptD, and dptE. Both NdeI / EcoRI restriction sites were used in the cloning primers, and the primers are shown in Table 2;

[0059] The genes of the four enzyme proteins were cloned by PCR technology, then digested with NdeI / EcoRI, and connected to the pET28-a (NOVAGEN) expression vector at the same restriction site, and then transformed with calcium chloride. The expression vector was transformed into the expression host E.coli BL21(DE3)plusS;

[0060] Induction: Pick a single colony of the transformant and inoculate it into 5 mL of LB liquid medium, and culture it on a shaker at 37°C overnight. On the second day, transfe...

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Abstract

The invention relates to an enzymatic synthesis method for DNA phosphorothioation. The enzymatic synthesis method for DNA phosphorothioation belongs to the technical field of biological medicines. The enzymatic synthesis method for DNA phosphorothioation comprises the following steps of DNA and L-cysteine as substrates are added with a DNA phosphorothioation modification zymoprotein or a DNA phosphorothioation modification zymoprotein-containing whole-cell lysate; and the mixture undergoes a phosphorothioation modification enzymatic reaction under suitable conditions to produce phosphorothioation-modified DNA. The enzymatic synthesis method for DNA phosphorothioation can realize controllable modification of DNA molecules having different length values and different sources. DNA modified by the enzymatic synthesis method has sequence specificity and spatial conformation specificity and can be used in fields of molecular biology research, gene therapy and gene vaccines.

Description

technical field [0001] The invention relates to the phosphorothioylation modification of DNA molecules, in particular to an enzymatic synthesis method for the phosphorothioylation modification of DNA molecules. Background technique [0002] Phosphorothioylated DNA can resist the action of various DNA hydrolases, so it has a wide range of applications in gene therapy and molecular biology research, such as gene blocking, site-directed mutagenesis, etc. At present, the phosphorothioylation modification of DNA requires chemical synthesis, but the chemical method has many limitations, such as low catalytic efficiency, only short chains can be synthesized, and phosphorothioylated DNA is a mixture of two optical isomers. Mixtures, complex purification and separation steps, etc. In addition to solving the problems encountered in chemical synthesis, the use of enzymatic methods for DNA phosphorothioylation modification has many advantages, such as the controllable modification of D...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12R1/42
Inventor 由德林曹博邓子新郑晓青
Owner SHANGHAI JIAO TONG UNIV
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