Product info: Bls I


Name
Bls I
Cat. #E533E534
Package, u.a.100500
Concentration, u.a./ml50005000

Recognition site
DNA sequence with at least two 5mC:
PuPyN↑PuPy
PyPu↓NPyPu
SourceBacillus simplex 23
Substrate specificityThe enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].

BlsI cleaves DNA sequence 5`- PuPyNPuPy-3`/3`-PyPuNPyPu-5', if at least two 5-methylcytosines are present in the recognition site (N isn`t considering).

The enzyme activity varies for different sites and depends on number and position of methylated cytosines in the recognition sequence.

Optimal recognition site (100% activity )
5`-G(5mC)NGC-3`/3`-(5mC)GN(5mC)G-5`.
Assayed onDNA pFsp4HI3/DriI is a linearized plasmid pFsp4HI3, which carries a gene of DNA-methyltransferase M.Fsp4HI and includes three canonical sites:
5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5` [2].
Unit definitionOne unit is defined as the amount of enzyme
required to hydrolyze at least one of three canonical sites
5`-G(5mC)NG(5mC)-3`/3`-CGN(5mC)G-5`
in 1 μg of linearized plasmid pFsp4HI3 in 1 hour at 30°C in a total reaction volume of 50 μl. As a result a linearized plasmid pFsp4HI3 disappears (see run 4 in the figure).
BlsI activity assay on DNA pFsp4HI3/DriI

Lanes:
1 and 7 1 Kb SE DNA-markers.
2 Control DNA pFsp4HI3/DriI,
3 0.5 μl Bls I (1/5)
4 1 μl Bls I (1/5)
5 2 μl Bls I (1/5)
6 1 μl of undiluted BlsI

Products were separated in 1,4% agarose gel in Buffer TAE.
BlsI activity assay on DNA pFsp4HI3/DriI
Optimal SE-bufferW (10 mM Tris-HCl (pH 8.5 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT.)
Enzyme activity (%)
BGOWYRose
10 - 2510 - 2550 - 7510075 - 10050
Reaction bufferSE-buffer W, (10 mM Tris-HCl (pH 8.5 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT.)
Optimal temperature
30oC
Storage conditions10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg /ml BSA; 50% glycerol; Store at -20°C.
Non-specific hydrolisisNo detectable degradation of 1μg of Lambda DNA was observed after incubation with 5 units of enzyme for 16 hours at 30°C in a total reaction volume of 50 μl.
Reagents Supplied with Enzyme 10 X SE-buffer W
Methylation sensitivityThe enzyme cleaves C5-methylated DNA and doesn't cut unmodified DNA [1].
Inactivation 20 minutes under
65oC
MSDS:Download MSDS as PDF
References:1. Chernukhin V.A., Tomilova J.E., Chmuzh E.V., Sokolova O.O., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Bacillus simplex - producer of BlsI site specific endonuclease. // Russian Federation patent RU 2322494 C1 (2006).
2. Chmuzh E.V., Kashirina Yu.G., Tomilova Yu.E., Chernukhin V.A., Okhapkina S.S., Gonchar D. A., Dedkov V.S., Abdurashitov M. A., Degtyarev S. Kh. Gene cloning, comparative analysis of the protein structures from Fsp4HI restriction-modification system and biochemical characterization of the recombinant DNA methyltransferase M.Fsp4HI. // Molecular Biology, V.41, No 1, p. 43-50 (2007)

Application:
 V.A. Chernukhin, Yu.E. Tomilova, E.V. Chmuzh, O.O. Sokolova, V.S. Dedkov, S.Kh. Degtyarev Site-specific endonuclease BlsI recognizes DNA sequence 5-G(5mC)N^GC-3 and cleaves it producing 3 sticky ends // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 1, pp 28-33, 2007
 Chernukhin V.A, Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Degtyarev S.Kh. Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 4, pp 19-27, 2007
 D. A. Gonchar, A. G. Akishev, S. Kh. Degtyarev BlsI- and GlaI-PCR assays a new method of DNA methylation study // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.6, No 1, pp 5-12, 2010
 Alexander G. Akishev, Danila A. Gonchar, Murat A. Abdurashitov and Sergey Kh. Degtyarev Epigenetic typing of human cancer cell lines by BlsI- and GlaI-PCR assays // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 3, pp 5-16, 2011