- Restriction enzymes.- For complette list,
please click here: Overview
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- Description
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Restriction enzymes are endonucleases that recognize specific ds DNA
sequences and cleave the DNA in both strands. Three classes are recognized:
Type I (EC 3.1.21.3.) cleave the DNA at apparently random sites far away
from the recognition site. Type III enzymes (EC 3.1.21.5.) cleave the
DNA outside of the recognition site. Most characterized enzymes belong
to the Type II class (EC 3.1.21.4); together with the Type IIS class they
comprise the commercially available restriction enzymes used for DNA analysis
and manipulation cleavage within the recognition site. Type II enzymes
(EC 3.1.21.4) recognize symmetric DNA sequences and cleave within the
sequences, leaving a 3´-hydroxyl on one side of the cut and a 5´-phosphate
on the other. Type IIS enzymes recognize asymmetric and uninterrupted
sequences, 4-7 bp in length. They cleave at a defined distance, up to
20 bp, to one side of their recognition sequence.
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- Unit definition
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1U is defined as the activity that cleaves 1 µg of lambda DNA
in a 50µl reaction in 1 h under optimum buffer conditions at optimum
temperature (usualy 37°C).
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- Reaction buffers
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- Each enzyme supplied with a 10x reaction buffer:
- Reaction buffer A (10X): 660 mM K-acetate, 330 mM Tris-acetate pH
7.9, 100 mM Mg-acetate, 5 mM DTT.
- Reaction buffer B (10X): 1 M NaCl, 100 mM Tris-HCl pH 8.0, 50 mM MgCl2,
10 mM 2-mercaptoethanol.
- Reaction buffer M (10X): 500 mM NaCl, 100 mM Tris-HCl pH 7.5, 100
mM MgCl2, 10 mM DTE.
- Reaction buffer L (10X): 100 mM Tris-HCl pH 7.5, 100 mM MgCl2,
10 mM DTE.
- Reaction buffer H (10X): 1 M NaCl, 100 mM Tris-HCl pH 7.5, 100 mM
MgCl2, 10 mM DTE.
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- Reaction conditions
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- The preparation of DNA to be cleaved should be free of contaminants
such as phenol, chlorophorm, alcohol, EDTA, detergents, or excessive
salts, all of which can interfere with restriction endonuclease activity.
The activity and specificity of some REs are influenced by the methylation
state of the DNA substrate. To avoid site-specific dam and/or dcm methylation
of recognition sites in E. coli vectors, it is necessary to propagate
the vectors in strains lacking one or both of the methyltransferase
systems. It must be taken into account, however, that E. coli
strains, deficient in dam or dcm methylation, have elevated rates of
mutation.
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- Star activity is defined as a relaxed specificity of restriction
enzymes under suboptimal reaction conditions (e.g. high glycerol concentration,
low ionic strength or high pH values in the reaction buffer) and is
related to the naturally observed limited accuracy of these enzymes
when used in high concentration over a prolonged time. To keep glycerol
concentration at less than 5% in a reaction, the restriction endonuclease
should not exceed 10% of the total reaction volume.
- Depending on the specific purpose, the digestion reaction is terminated
by inhibiting, destroying or extracting the restriction enzyme. This
can be achieved by chelation of the essential cofactor Mg2+,
thermal denaturation (usualy, at 65° to 95° for 10 min), or
by phenol/chloroform extraction.
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- Storage
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- Store at -20°C.
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- Applications
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- Applications: analysis of the methylation state of genes (e.g., Hpa
II and Msp I); RFLP analysis; genetic fingerprinting; cloning, restriction
mapping, DNA labeling.
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Quality control
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activity, purity, absence of nonspecific endonucleases/exonucleases/other
specific restriction endonucleases/phosphatases, ligation-recut assay.
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References
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1. http://rebase.neb.com
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