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.Copyright © Suzhou Yacoo Science Co., Ltd. All Rights Reserved
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