Application of Dithiothreitol|CAS:3483-12-3| in Enhancing the Activity of CELI Family Enzymes

CELI enzyme is a specific nuclease that specifically recognizes both single-stranded and double-stranded. At present, CELI enzyme is mainly extracted from celery. This enzyme can specifically cleave mismatched bases and is widely used in the field of mutation detection. Traditional theories believe the catalytic activity of the enzyme only requires Mg²⁺ and Zn²⁺. Dithiothreitol, English name DL-Dithiothreitol, abbreviated as DDT, molecular formula is C₄H₁₀O₂S₂, molecular weight is 154.25, often used as a reducing agent, has antioxidant effect. Today I will introduce the application of DTT in the preparation of CELI family enzyme digestion buffer.

The CELI family enzyme digestion buffer is composed of DTT, BSA (bovine serum albumin), 0.02% Triton X-100, 0.1M KCl and HEPES buffer, among which:

The CELI family enzyme digestion buffer containing DTT is composed of        Mutation Detection Kit-S100 kit buffer and DTT.  Mutation Detection Kit-S100 kit buffer is a product of Universal Gene Corporation, the catalog number is 706025;

The concentration of DTT in CELI family enzyme digestion buffer is 0.01-0.5mM, 0.5-10mM or 0.01-6mM;

The BSA concentration is 2ug/ml;

The concentration of HEPES buffer is 0.1M and the PH is 7.5.

The new catalytically active substance DTT can further improve the activity of CELI, especially for the detection of low-abundance mutations. Compared with the traditional enzyme digestion method, the method of the present invention is more sensitive, and the final concentration of DTT is in the range of 0.01-10 mM. Both can improve the detection sensitivity of CELI enzyme and CELII enzyme. The detection of low-abundance unknown mutants, such as a certain cancer gene, has a good detection effect. Especially when the TILLING technology platform detects mutagenic populations, it has obvious advantages. It not only simplifies the PCR purification steps, but also improves Detection resolution.

References

CN106191228A Application of DTT in improving CELI family enzyme activity.