|Gla I|| |
|Cat. #||E493||E494 |
|Package, u.a.||100||500 |
|Concentration, u.a./ml||10000||10000 |
|Source||Glacial ice bacterium GL29|
|Substrate specificity||The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA
and DNA with N4-methylcytosines .|
The enzyme activity depends on number and position of methylated nucleotides in the recognition sequence :
Optimal substrate (100% activity )
Good substrates ( > 25% activity)
Medium substrates ( > 6% activity)
Bad substrates (6% activity)
|Assayed on||DNA pHspAI2/GsaI is a linearized plasmid pHspAI2, which carries a gene of DNA-methyltransferase M.HspAI (recognition sequence 5`-GCGC-3`) and includes a unique GlaI recognition site|
|Unit definition||One unit is defined as the amount of enzyme required to completely digest a unique 5`-G(5mC)G(5mC)-3`/3`-(5mC)G(5mC)G-5` site in 1 μg of pHspAI2 plasmid DNA, which is linearized with GsaI, in 1 hour at 30°C in a total reaction volume of 50 μl. As a result of this site hydrolysis two main DNA fragments (3,419 and 699 bp) are produced (see lanes 3-5 in the figure). GlaI digestion of recognition sequences with three and two 5-methylcytosines results in additional bands appearance (lane 6 in the figure).
|GlaI activity assay on DNA pHspAI2/GsaI|
2 Control pHspAI2/GsaI DNA
3 - 0.5 μl GlaI (diluted 1/20),
4 - 1 μl GlaI (diluted 1/20),
5 - 2 μl GlaI (diluted 1/20),
6 - 1 μl of undiluted GlaI,
1 and 7- 1 Kb SE DNA Ladder.
Products were separated in 1% agarose gel in TAE Buffer.
|Optimal SE-buffer||Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)|
|Enzyme activity (%)|
|75 - 100||75 - 100||25 - 50||25 - 50||100||100|
|Reaction buffer||SE-buffer Y, (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.)|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,05% Triton X-100, 0.1 mg/ml BSA, 50% glycerol; Store at -20°C.|
|Non-specific hydrolisis||No detectable degradation of 1μg of Lambda DNA was observed after incubation with 8 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 Y, pHspAI2/GsaI DNA|
|Methylation sensitivity||The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNA
and DNA with N4-methylcytosines .|
|Inactivation 20 minutes under||65oC|
|MSDS:||Download MSDS as PDF|
|References:||1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh. Bacterial strain Glacial ice bacterium I - producer of GlaI restriction endonuclease. // Russian Federation patent RU 2287012 C1 (2006).|
2. Valery A. Chernukhin, Tatyana N. Najakshina, Murat A. Abdurashitov, Julia E. Tomilova, Nina V. Mezentzeva, Vladimir S. Dedkov, Natalya A. Mikhnenkova, Danila A. Gonchar, Sergei Kh. Degtyarev A novel restriction endonuclease GlaI recognizes methylated sequence 5’-G(m5C)^GC-3’ // Biotechnologia (russ.). 2006. N 4. P. 31-35
3. Tomilova J.E., Chernukhin V.A., Degtyarev S.Kh. Dependence of site-specific endonuclease GlaI activity on quantity and location of methylcytosines in the recognition sequence 5’-GCGC-3’. // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.2, No 1, pp 30-39 (2006) (Russian)
4. G. V. Tarasova, T. N. Nayakshina, S. Kh. Degtyarev Substrate specificity of new methyl-directed DNA endonuclease GlaI // BMC Molecular Biology 2008, 9:7
5. Zemlyanskaya, E.V., Degtyarev, S.K. Substrate specificity and properties of methyl-directed site-specific DNA endonucleases.// Molecular Biology, v.47, No.6, p.900-913 (2013)
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
Abdurashitov M.A., Chernukhin V.A, Gonchar D.A., Degtyarev S.Kh.
GlaI digestion of mouse γ-satellite DNA: study of primary structure and ACGT sites methylation // BMC Genomics 2009, 10:322.
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
Wood, R. J., McKelvie, J. C., Maynard-Smith, M. D., and Roach, P. L. A real-time assay for CpG-specific cytosine-C5 methyltransferase activity.// (2010) Nucleic Acids Res 38, e107
Kravets AP, Mousseau TA, Litvinchuk AV, Ostermiller Sh, Vengen GS, Grodzinski DM. Changes in wheat DNA methylation pattern after chronic seed gamma-irradiation.// Tsitol Genet. 2010 Sep-Oct;44(5):18-22. Russian.
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
F. Syeda, R.L. Fagan, M. Wean, G.V. Awakumov, J.R. Walker, S. Xue, S. Dhe-Paganon, & C. Brenner, "The RFTS Domain is a DNA-competitive Inhibitor of Dnmt1"//, JBC, v. 286, pp. 15344-15351 (2011).
Keith N. Rand, Graeme P. Young, Thu Ho and Peter L. Molloy
, "Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylationdependent restriction enzymes."//, Nucleic Acids Research, pp. 1-10 (2012).
Fagan RL, Wu M, Chedin F, Brenner C
An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes //PLoS ONE 8(11): e78752. doi:10.1371/journal.pone.0078752 (2013)
Kuznetsov V.V., Abdurashitov M.A., Akishev A.G., Degtyarev S.H. Method for detection nucleotide sequence R(5mC)GY in given position of continuous DNA.// Russian Federation patent RU 2525710 C1 (2014).