RESTRICTION ENZYMES
Objectives
1) To know common problems related to the use of restriction enzymes and how to detect and overcome
them.
2) To be able to evaluate the accuracy of sizing DNA fragments on an electrophoretic gel.
3) To know what a Southern Blot is.
4) To know about infrequently cutting restriction endonucleases.
General References:
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Geoffrey G. Wilson, Noreen E. Murray (1991) Ann. Rev. Genet. 25: 585-627.
Robert Yuan (1981) Ann. Rev. Bioch. 50: 285-315
Every restriction enzyme manufacturer supplies a wealth of information about its enzymes in its
catalogue and in specification sheets supplied with the enzymes.
A running compendium of all available restriction enzymes is maintained at
http://rebase.neb.com/rebase/.
Background about restriction enzymes that you are expected to have:
 Type I restriction enzymes K & B as an impediment to cloning, and alternative designations of
them (ie. hsdR, hsdM, hsdS, rK, mK)
 Type II enzymes (like EcoRI), use in cloning, interaction with methylases, isoschizomers,
types of ends (blunt, 5' recessed, 3' recessed), compatible ends.
 Dephosphorylation of ends as a means of preventing ligation.
 Use of synthetic linkers.
 Expected frequency of 4 base sites, 6 base sites, etc.
Modern purposes of restriction digestion
 Until recently, new clones were often subjected to restriction mapping by double digestion as a
means of sorting out which features were on which fragments and merited what kinds of
characterization. More recently, clones are usually of known sequence, so inferences from
sequence searches are used to answer those kinds of questions.
 However, it is important to measure restriction patterns for clones and compare them to the
patterns expected from the sequence. This reveals rearrangements that happen during clone
growth, mislabeled clones, and sometimes errors in the original sequence assembly.
 Of course restriction enzymes are still used in clone construction. But clone construction often
is facilitated with PCR tricks that place fairly light demands on presence of restriction sites.
 One of the most important purposes of restriction digestions is to verify that a clone has been
properly constructed. One of the most common problems encountered is for an unexpected
stray fragment to get ligated into a clone in addition to the targetted insert. Similarly,
restriction digestion is often a convenient way to determine the orientation of an insert.
Practical aspects of using restriction enzymes (Type II restriction endonucleases).
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Problems related to not enough activity:
 Restriction enzymes, like other enzymes, have an activity defined by cleaving a certain amount of
substrate to completion in a specified time and under specified conditions. Typically a "unit" is
defined as cleaving 1 ug of DNA per hour under optimal reaction conditions. Usually the vendor
supplies a 10x buffer concentrate for composing your reactions in the optimal conditions. The "1
ug of DNA" contains a trap in that it is the concentration of sites, not the concentration of
nucleotides that matters. For example, EcoRI is typically assayed on lambda DNA that has 1 site
per 10 kb. You might use it on the PCR product of 100 bp having 1 site engineered into each
primer. You will need 200 x as much enzyme as you might have expected.
 Typically, one plans for 5-10 times more activity than calculated from the advertised units to cover
various causes of slow cleavage. These include certain sites that intrinsically cleave slowly,
suboptimal conditions (carry over of salt, wrong pH, carry over of competing polyanions like
tRNA, etc), misestimated DNA concentration, sites too close to the ends of a fragment, and
enzyme preparations that have partially lost activity. If at all possible, one should digest, then
withdraw some material to check on a gel, and have enough left over to digest some more, check
on a gel again, and still have enough to do the planned experiment.
 The amount of activity applied can be increased by increasing time instead of just adding more
enzyme. The "fold excess" should be calculated as hours times units/ug (adjusted for
concentration of sites if appropriate). This, of course, assumes that the enzyme will survive long
times under reaction conditions. Stability varies by enzyme, and by the amount of other protein
present. One could hope that the manufacturer's specifications sheet that comes with each enzyme
would warn if the enzyme is particularly unstable. Long term stability of nearly all enzymes
benefit by the presence of carrier protein. Manufacturers generally supply BSA and recommend 1
ug/ul for stabilizing the enzymes. Another good option is 0.5 ug/ul of gelatin. Gelatin can be
autoclaved, so it is a good choice if contamination with stray activities is a concern. Reactions of
duration greater than over night should be supplemented with a small amount of sodium azide to
prevent bacterial growth.
Problems related to too much activity.
 Usually one cannot add more than 10% enzyme stock solution to a reaction without the enzyme
storage buffer disturbing the reaction conditions. A common phenomenon is to encourage relaxed
specificity due to agents like glycerol in the storage buffer. This is sometimes called "star"
activity.
 Commercial restriction enzymes are not pure, and are generally contaminated with non specific
nucleases and phosphatases. Extensive overdigestion typically first damages DNA ends so that the
fragments will not ligate, and eventually destroys the integrity of the fragments all together.
Exonucleolytic damage produces the paradoxical effect that the fragments appear to smear
upwards on electrophoretic gels. This is because single stranded DNA migrates slower than
double stranded DNA. Most commercial enzymes are OK at 100 fold over digestion, but not at
1000 fold over digestion. Particularly contaminated enzymes should be ascertainable from the
technical specifications sheet provided by the manufacturer. If they say they got complete
digestion and > 95% religation at 10 fold overdigestion, that means that it was terrible at 100 fold
overdigestion.
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Troubleshooting.
 If a target DNA will not cleave, then test the enzyme on a clean control substrate. If the enzyme is
OK on its control, then mix experimental and control DNA together and try to cleave. If the
experimental DNA causes the control to not cleave, your problem is purity of the experimental
DNA. If the control cleaves while the experimental DNA does not, then the problem is with the
covalent nature of the experimental DNA. Either it has no sites, or they are modified (eg. by
methylation). In the case of modification, you may be able to find enzymes that cleave the same
site but have a different sensitivity to modification. You may also remove modifications by
conducting in vitro replication through PCR.
Assay:
 Most typically restriction digests are assayed by electrophoresis on a small gel of 0.7 to 1%
agarose. This technique is fast and simple. Although polyacrylamide gels have higher resolution
for fragments below 500 bp, most people try to get by with higher percentage agarose, or certain
brands of agarose engineered for resolution of smaller fragments. Capillary gel electrophoresis is
also possible, but the equipment available within the department is configured for fluorescently
labelled fragments, and is only applicable to special applications.
 Visualization: Although other staining agents are available, most commonly the DNA is
visualized by staining in 0.5 ug/ml ethidium bromide, which can either be soaked into the gel or
placed in the gel during its original formation. Visualization requires illumination with a UV light.
A wave length of 265 nm is most effective, but also does more damage to the DNA and is a safety
concern. Illumination at 300 nm is common, particularly if the DNA is to be recovered from the
gel for cloning. For imaging the gel may be either illuminated from below or above. The
investigator should never stare into a gel laying on top of a UV light source, at least not without a
UV face shield. There are a number of high tech imaging devices ranging from specialized gel
imagers to home-made CCD cameras mounted on laboratory computers. Many of us still use the
ancient Poloroid camera setup in the C corridor common room. Sensitivity typically allows
visualization down to 5-10 ng in a band. One should always record an image of every gel.
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The experiment depicted above is suboptimal for a number of reasons. Can you see why?
There should be more markers, and there should be markers extending below the lowest
experimental band of interest and markers better defining the onset of curvature. The gel could
have been run longer to make better use of its revolving power.
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The major problem in using restriction fragment sizes for any purpose is the likelihood of
confusing two fragments that are coincidentally close in size. This problem increases dramatically
with the degree of sizing error and the numbers of fragments in the digest. Sizing error can be
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evaluated by the degree of deviation from a smooth curve within the standard, and by the deviation
from the known size of some fragment in a second lane. The vector is often used for the latter.
Error > 5% is due to poor technique. Accuracy down to 3% can be achieved with standards and
unknowns loaded in the same lane to remove lane-to-lane variability. Lane-to-lane variability can
be due to differing concentrations of salt in the different samples. Below 3% uncertainty, the base
composition of fragments, and the nature of the ends may become a factor, particularly on small
fragments.
Understanding circular DNA
Plasmid DNA as isolated from cells is a mixture of forms: monomer covalently closed
circle (ccc) (supercoiled), dimer and higher forms of ccc, and nicked circles (alias relaxed circles).
The supercoils generally run as if they were about half the size of the corresponding linear,
although the relationship between ccc and linear migration is dependent on gel conditions. The
sizing curve for circles remains linear higher up on the gel.
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Quantitation of clones using restriction digests:
 A restriction digest of some standard molecule, such as bacteriophage lambda DNA, will serve as
both a size marker and a quantitation standard curve. Each fragment is present in an amount in
micrograms that is in proportion to its size in bp. Either by eye, or by a quantitative scan, one may
quantitate an unknown against the resulting standard curve. Be careful about saturation. For
example, a photographed band is often white. This is saturated, meaning that the film can't get any
whiter. So one can't compare two saturated bands and know which one was "brighter".
Intuitively, people tend to observe the band width in this case. But beware that bands also get
broader for smaller fragments. Paying attention to the quantitation after digestion where there are
bands of several different sizes to judge often gives the first warning that the initial estimate of
DNA quantity was way off.
Infrequently cutting enzymes for mammalian DNA:
NotI
BssHII
MluI
NruI
SfiI
SalI
ClaI
GC^GGCCGC
G^CGCGC
A^CGCGT
TCG^CGA
GGCCN4^NGGCC
G^TCGAC
AT^CGAT
~4x106
~3x105
~3x105
~3x105
~7x104
~7x104
~7x104
Infrequently cutting enzymes are sometimes included in cloning vectors as a site for recleavage
that is unlikely to be present in the insert.
Infrequently cutting enzymes are sometimes used for examination of genomic DNA to get a broad
scale verification by restriction mapping of a sequence assembly, or to investigate the distribution
of CG modifications. CG is more rare in mammalian DNA than one would expect in random
sequence. The frequency of CG is depressed in mammalian DNA because methylation at the C
promotes mutation to a T by deamination. Also, many of the sites are not cleaved because they
are methylated. Most of the unmethylated CGs in the genome are clustered in "CG islands".
Examination of large restriction fragments requires a special electrophoresis system called "pulsed
field electrophoresis". In ordinary gel electrophoresis, small DNA fragments migrate as a random
coil reflecting the length of the molecule. For large fragments, the random coil elongates in the
direction of migration, such that increased size ceases to be a factor in its migration. In pulsed
field electrophoresis, the direction of the voltage is frequently shifted to prevent this effect. There
are several versions of the method. To get very large fragments into the gel without shearing them,
the cells are often placed intact in an agarose plug, and lysed and cleaved in situ.
For visualization of individual fragments within the context of an entire digested genome, a
hybridization method called a Southern Blot is used.
Southern blotting
Alternatives: Southern blotting is nearly always substituted by some combination of PCR and
DNA sequencing whenever possible these days. One common use remaining is to characterize transgenes
in transgenic animals, particularly in untargetted constructs.
Southern blotting is used to tell which bands on a gel share sequences with some hybridization
probe. The DNA in the gel is denatured with alkali, and then transferred onto a piece of filter paper by
one of a variety of methods. The hybridization probe is radiolabeled, denatured, and allowed to reanneal
with the DNA affixed to the filter. The filter is washed under conditions that remove probe that is not
annealed to DNA on the filter, and then the filter is subjected to autoradiography.
If the probe is not perfectly matched to the target, or if there are related sequences that cross
hybridize in the clone, then the washing conditions can be adjusted to try to include or exclude hybrids
with some particular degree of mismatching. The degree of tolerance to mismatching is called
"stringency".
Stringency
High
Moderate
Low
Mismatch tolerated
0 - 8%
0 - 20%
0 - 30%
Temp.(in 1.5 mM Na+)
62
52
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The washing is a kinetic experiment, not an equilibrium one. The above conditions completely
remove the signal from hybrids below stringency in about an hour of washing. The wash buffer
should be frequently changed to keep reannealing from occurring.
Last revised 2/21/05 - Steve Hardies