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商品編號


R0662S-200 units



品牌


紐英倫(NEB)



公司


New England Biolabs



公司分類(lèi)


Restriction Endonucleases



Concentration

5,000 units/ml


Size

200 units

商品信息

Description:
FspEI, an EpiMark
?
validated product, is a modification-dependent endonuclease which recognizes C
m
C sites and generates a double-stranded DNA break on the 3? side of the modified cytosine at N
12
/N
16
. Recognized cytosine modifications include C5-methylation (5-mC) and C5-hydroxymethylation (5-hmC) (1).?
This enzyme is provided with an Enzyme Activator Solution which may be used for efficient digestion by FspEI.
The most common epigenetic modifications found in eukaryotic organisms are methylation marks at CpG or CHG sites. A subset of these modified sites are recognized and cleaved by FspEI.?
At fully methylated CpG sites:?
5?. . . C
m
C? G G . . . 3?
3?. . . G? G
m
C C . . . 5?
or CHG sites:?
5?. . . C
m
C H? G G . . . 3?
3?. . . G? G D
m
C C . . . 5?
H = A or C or T (not G)
D = A or G or T (not C)?
FspEI recognizes each hemi-methylated site individually and cleaves bidirectionally to generate 32 base or 31 base fragments, respectively. These fragments contain the central methylated site and have 4-base 5? overhangs at each end. FspEI does not cleave unmodified DNA.
Product Source
An?
E. coli?
strain that carries the synthetic FspEI gene from?
Frankia
?species EAN1pec.
Reagents Supplied
The following reagents are supplied with this product:
Store at (°C)
Concentration
CutSmart? Buffer
-20
10X
Enzyme Activator Solution
30X
Notes:
This enzyme has been used for epigenetic analysis in which target DNA containing CpG sites methylated in both strands is cleaved by FspEI to generate 32 bp fragments containing the methylated CpG sites. The isolated 32 bp fragments can be sequenced to reveal 5-mC modification sites (Cohen-Karni et al., (2011) PNAS 108: 11040-11045).Use of excess enzyme inhibits cleavage. Optimization of the amount of enzyme needed for complete digestion may be required for each substrate DNA.Star activity, including digestion of non-methylated DNA substrates, may result from non-optimal reaction conditions such as extended digestion time, high enzyme or activator concentration or a glycerol concentration of >5%. Optimization of the amount of enzyme needed to achieve efficient specific cuffing, while avoiding star activity, may be required for each substrate DNA.Optimal specificity of cleavage at 5-mC sites can be achieved by keeping the molar ratio of FspEI to 5-mC target sites between 0.1:1 to 1:1 in the presence of 1X Enzyme Activator Solution and digestion for up to 60 minutes. (The molarity of FspEI~0.6 μM).