{"id":1758,"date":"2022-04-04T22:03:00","date_gmt":"2022-04-04T15:03:00","guid":{"rendered":"http:\/\/sibenzyme.com\/product\/gla-i\/"},"modified":"2026-01-26T18:03:53","modified_gmt":"2026-01-26T11:03:53","slug":"gla-i","status":"publish","type":"product","link":"https:\/\/sibenzyme.com\/en\/product\/gla-i\/","title":{"rendered":"Gla I"},"content":{"rendered":"

Recognition site and hydrolysis position<\/strong>:<\/p>\n\n\n\n\n
R(5mC)GY<\/td>\nR(5mC)\u2191GY<\/td>\n<\/tr>\n
YG(5mC)R<\/td>\nYG\u2193(5mC)R<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Source:<\/strong> An E.coli strain that carries the cloned Gla I gene from Glacial ice bacterium Gl29
\nSubstrate specificity<\/strong>:
\nUnit definition: <\/strong>One unit is defined as the amount of enzyme required to hydrolyse completely a unique 5`-G(5mC)G(5mC)-3`\/3`-(5mC)G(5mC)G-5` site in 1 \u03bcg of pHspAI2 plasmid DNA, which is linearized with GsaI, in 1 hour at 30\u00b0C in a total reaction volume of 50 \u03bcl. 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).<\/p>\n\n\n\n
GlaI activity assay on DNA pHspAI2\/GsaI<\/b>
\nLanes:
\n2 Control pHspAI2\/GsaI DNA
\n3 – 0.5 \u03bcl GlaI (diluted 1\/100),
\n4 – 1 \u03bcl GlaI (diluted 1\/100),
\n5 – 2 \u03bcl GlaI (diluted 1\/100),
\n6 – 1 \u03bcl of undiluted GlaI,
\n1 and 7- 1 Kb SE DNA Ladder.Products were separated in 1% agarose gel in TAE Buffer.<\/td>\n		\"GlaIIn the presence of 20% DMSO, the enzyme’s activity significantly increases without losing specificity<\/span><\/a><\/mark><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Assayed on: <\/strong>DNA pHspAI2\/GsaI<\/b><\/a><\/span> is a linearized plasmid pHspAI2<\/b><\/a><\/span>, which carries a gene of DNA-methyltransferase M.HspAI <\/b><\/a><\/span> (recognition sequence 5`-GCGC-3`) and includes a unique GlaI recognition site 5`-G(5mC)G(5mC)-3`\/3`-(5mC)G(5mC)G-5` [2]<\/span><\/a>.
\nOptimal SE-buffer:<\/strong> SE-buffer Y
\nEnzyme activity (%)<\/strong>:<\/p>\n\n\n\n\n
B<\/td>\nG<\/td>\nO<\/td>\nW<\/td>\nY<\/td>\nROSE<\/td>\n<\/tr>\n
75<\/td>\n75<\/td>\n25<\/td>\n25<\/td>\n100<\/td>\n100<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Storage conditions<\/strong>: 10 mM Tris-HCl (pH 7.6); 250 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 0,1% Triton X-100, 0.05 mg\/ml BSA, 50% glycerol; Store at -20\u00b0C.
\nLigation<\/strong>: –
\nNon-specific hydrolisis<\/strong>: No detectable degradation of 1\u03bcg of Lambda DNA was observed after incubation with 100 units of enzyme for 16 hours at 30\u00b0C in a total reaction volume of 50 \u03bcl.Reagents Supplied with Enzyme<\/b><\/p>\n

10 X SE-buffer Y, pHspAI2\/GsaI DNA
\nMethylation sensitivity<\/b>: The enzyme cleaves only C5-methylated DNA and does not cut unmodified DNAand DNA with N4-methylcytosines [1]<\/a>.
\nNotes: <\/strong><\/p>\n

References<\/b>:<\/strong><\/p>\n

    \n
  1. Chernukhin V.A., Nayakshina T.N., Tomilova J.E., Mezentseva N.V., Dedkov V.S., Degtyarev S.Kh.<\/i><\/b> Bacterial strain Glacial ice bacterium I – producer of GlaI restriction endonuclease. \/\/ Russian Federation patent RU 2287012 C1 (2006).<\/li>\n
  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<\/strong> A novel restriction endonuclease GlaI recognizes methylated sequence 5\u2019-G(m5C)^GC-3\u2019 \/\/ Biotechnologia (russ.). 2006. N 4. P. 31-35<\/a><\/li>\n
  3. Degtyarev S.Kh., Belavin P.A., Repin V.E., Malygin E.G.<\/strong> Preparation method of restriction endonuclease Fok I from Flavobacterium okeanokoites \/\/ Soviet Union patent SU1436160 (1988). (In Russian)<\/li>\n
  4. Degtyarev S.Kh., Rechkunova N.I., Grinev A.A., Dedkov V.S.<\/strong> Discovery and substrate specificity determination of restriction endonucleases Bme18I and Kzo9I.\/\/ Izvestia SB SA USSR, Biological sciences series, No.3, 25-26 (1989). (In Russian)<\/li>\n
  5. Zemlyanskaya, E.V., Degtyarev, S.K.<\/i><\/b> Substrate specificity and properties of methyl-directed site-specific DNA endonucleases.\/\/ Molecular Biology, v.47, No.6, p.900-913 (2013)
    \n<\/b><\/li>\n<\/ol>\n

    Application:<\/b><\/p>\n