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Restriction Enzymes

Restriction enzymes were first purified from bacteria over 30 years ago following

investigations into the host-specific restriction and modification of bacterial viruses.

Among the first enzymes purified that were responsible for this phenomenon were the

type II enzymes EcoR I from Escherichia coli and Hind III from Haemophilus

influenzae. Tens of thousands of bacteria and archae have been screened for restriction

enzymes, among these, nearly 3000 enzymes have been found, exhibiting over 200

different specificities. It is generally believed that the biological function of restriction

enzymes is to protect cells from foreign DNA: infecting DNA is cleaved preventing it

from successfully replicating and parasitizing the cell. Usually, organisms that make

restriction enzymes also make a companion modification enzyme (DNA

methyltransferase) that protects their own DNA from cleavage. These enzymes

recognize the same sequence as the restriction enzyme the accompany, but instead of

cleaving it, they disguise it by methylating one of the bases in each DNA strand.

Together, a restriction enzyme and its cognate modification methyltransferase form a

so-called restriction-modification (R-M) system. At least 4 different kinds of R-Msystems exist; most characterized enzymes (93%) belong to the type II class.

The type II enzymes are the simplest: they recognize symmetric DNA sequences and

cleave within the sequences, leaving a 3-hydroxyl on one side of the cut and a 5-

phosphatase on the other. They require only magnesium for activity and their

corresponding modification enzymes require only S-adenosyl-methionine. The variety

of sequences recognized is virtually unlimited, though few contain less than four or

more than eight specific bases. Together with the type IIS class (5%) they comprise the

commercially available restriction enzymes used for DNA analysis and manipulation.

All enzymes used in this course were obtained from New England Biolabs, Inc. (NEB)

[www.neb.com]. Along with the restriction enzymes a buffer is supplied (NEB1,2,3 or 4

or enzyme specific). A typical restriction digest follows the general rule that 10 units ofa restriction enzyme is sufficient to overcome variability in DNA source, quantity and

purity (by definition, 1 Unit will completely digest 1g of DNA in a 50 l volume in 1

hour at the recommended temperature). Generally, 1 l of enzyme is added to 1 g of

purified DNA in a final volume of 20-50 l of 1x NEBuffer followed by incubation for

1 hour at the recommended temperature. If more enzyme is used, the length of

incubation can often be decreased to save time. Alternatively, you can productively

digest with fewer units of enzyme for up to 16 hours with many restriction enzymes.

Restriction enzymes should be kept on ice when they are not in the freezer. They should

always be the last component to be added, and the DNA to be cleaved should be free of

contaminants such as phenol, chloroform, EDTA, detergents, or excess salts, all of

which can interfere with the restriction enzyme activity.

NEB provides 10x NEBuffers which should be used at a 1x concentration in the

reaction. Some restriction enzymes require bovine serum albumin (BSA) at a final

concentration of 100 g/ml for optimal activity. Restriction enzymes that do not require

BSA are not adversely affected if it is present in the reaction. If no further

manipulations of the digested DNA are planned, the reaction can be stopped by adding a

stop solution (e.g. 50% glycerol, 50 mM EDTA, and 0.05% bromophenol blue

(10l/50l). If further manipulations are required, heat inactivation (e.g. 65C, 20 min)

is the simplest method.

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BamH I 5-G/GATCC-3

3-CCTAG/G-5

Buffer/Temp NEBufferBamHI (150 mM NaCl, 10 mM Tris-HCl, 10 mM

MgCl2, 1 mM dithiotreitol (pH 7.9 @ 25C) + BSA [37C]

NEB1: 75%, NEB2: 100%, NEB3: 50%, NEB4: 75%

EcoR I 5-G/AATTC-33-CTTAA/G-5

Buffer/Temp NEBufferEcoR I (50 mM NaCl, 100 mM Tris-HCl, 10 mM

MgCl2, 0.025% Triton X-100 (pH 7.5 @ 25C) [37C]

NEB1: 100%, NEB2: 100%, NEB3:100%, NEB4:100%

Hind III 5-A/AGCTT-3

3-TTCGA/A-5

Buffer/Temp NEB2 [37C]

NEB1: 50%, NEB2: 100%, NEB3: 10%, NEB4: 50%

Nco I 5-C/CATGG-3

3-GGTAC/C-5

Buffer/Temp NEB4 [37C]

NEB1: 100%, NEB2: 100%, NEB3:100%, NEB4:100%

XbaI 5-T/CTAGA-3

3-AGATC/T-5

Buffer/Temp NEB2 + BSA [37C]

NEB1: 0%, NEB2: 100%, NEB3:75%, NEB4:75%

CHECK OUT FOR MORE: www.neb.com

1xNEB1: 10 mM Bis Tris Propane HCl, 10 mM MgCl2, 1 mM dithiotreitol (pH 7.0)

1xNEB2: 10 mM Tris-HCl, 50 mM MgCl2, 50 mM NaCl, 1 mM dithiotreitol (pH 7.9)

1xNEB3: 50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM dithiotreitol (pH 7.9)

1xNEB4: 20 mM Tris-Ac, 10 mM MgAc2, 100 mM NaCl, 1 mM dithiotreitol (pH 7.9)

DOUBLE DIGEST: If no single NEBuffer can be found to satisfy the buffer

requirements for both enzymes, the reactions can be done sequentially. First cleave with

the enzyme that requires the lower salt reaction conditions, then adjust the salt

concentration of the reaction and add the second enzyme.