Restriction Enzyme called the restriction site.

Most restriction
enzymes recognize a four to six base sequence that
Analysis is palindromic, meaning that both DNA strands
have the same sequence when the strands are read
from the 5’ to the 3’ direction (Figure 1).
Introduction
Once the recognition site is found, the restriction
Restriction enzymes (also known as restriction enzyme will catalyze the hydrolysis of a
endonucleases) act as molecular scissors to cut phosphodiester linkage on each strand of the
double-stranded DNA to produce double-stranded DNA, producing two DNA fragments, each with
fragments. These enzymes are made by bacteria as a phosphate group at the 5’ end and a hydroxyl
a mechanism of defense against viral infections. group at the 3’ end. Some restriction enzymes
Certain bacterial viruses (bacteriophages) infect cut both DNA strands in a symmetrical fashion
bacteria by inserting viral DNA into the bacterial within the recognition sequence. A symmetrical
cell. This DNA can then be replicated and cut of the recognition sequence generates DNA
transcribed by the host cell machinery, resulting fragments with blunt or flush ends (Figure 1).
in the production of new viruses and the eventual Other restriction enzymes make a staggered cut,
lysis of the cell. The bacterium’s restriction cleaving each strand off center in the recognition
enzymes will identify the foreign DNA and cut site. This produces DNA fragments with exposed,
it into many pieces, thus preventing replication single-stranded, “overhanging” 5’ and 3’ ends.
of the phage DNA. Restriction enzymes were so These fragments are commonly referred to as
named due to this ability to restrict, or limit, the cohesive or sticky ends.
number of viruses that can infect a bacterium.
For example, the restriction enzyme EcoRI
How do the restriction enzymes recognize and cleaves the DNA strand within the recognition
cleave only foreign DNA, and not the DNA site to produce a 5’ overhang of four nucleotides
from the bacterium’s own genome? The enzyme on each fragment that can serve as a template for
recognition sites within a bacterial cell’s genome base pairing with the complementary sticky ends
are protected from self-digestion by methylation of another DNA fragment cleaved by the same
of adenine or cytosine residues within those enzyme (Figure 1). Importantly, regardless of
sites. Because DNA methylation is a normal post- the source of DNA (whether from a prokaryote
synthesis DNA modification in many organisms, or eukaryote), EcoR1 will always cut at the same,
the foreign DNA will likely also have methylated specific recognition sequence and will always
bases. However, the methylation patterns in the produce restriction fragments with the same sticky
foreign DNA should vary enough from that of the ends that can base pair with another fragment that
host DNA to allow for its recognition and cleavage was digested with EcoR1.
by the restriction enzymes while keeping the host
DNA protected. The presence of methylation does The frequency that a restriction site is likely to
not, however, always protect DNA from restriction,occur within a DNA molecule can be estimated
as some restriction enzymes can recognize sites using statistical probability calculations. If we
that contain methylated bases. assume that the four nucleotides are distributed
randomly within a DNA molecule, then at any
A. Restriction Sites and Cleavage position in the molecule there is a 1/4 chance that
Products any of the 4 nucleotides will occupy that position.
Thus, any given four base-pair recognition site
A restriction enzyme functions by scanning DNA sequence will occur once every 256 bases (1/4 x
molecules for a specific sequence of nucleotides, 1/4 x 1/4 x 1/4 = 1/44 = 1/256). A specific six
 5' NNNNNNGGCCNNNNNNNNNNN 3' HaeIII 5' NNNNNNGG CCNNNNNNNNNNN 3'
3' NNNNNNCCGGNNNNNNNNNNN 5' 3' NNNNNNCC GGNNNNNNNNNNN 5'
blunt ends

5' NNNNNNGAATTCNNNNNNNNNN 3' EcoRI 5' NNNNNNG AATTCNNNNNNNNNN 3'

3' NNNNNNCTTAAGNNNNNNNNNN 5' 3' NNNNNNCTTAA GNNNNNNNNNN 5'
sticky ends

Figure 1. Restriction enzymes recognize specific 4-6 base pair sequences in DNA. The restriction en-
zyme HaeIII produces a symmetrical cleavage of the phosphate backbone within the restriction site to
create two blunt-ended fragments. EcoRI cleaves assymetrically within its recognition site, producing
two fragments with sticky ends. N represents any of the 4 nucleotides (A, T, C, or G) that may occupy
each position outside of the highlighted restriction site.
base-pair recognition site will occur, on average, For example: EcoRI (pronounced “eeko-are-
once every 4,096 bases (1/4 x 1/4 x 1/4 x 1/4 x one”)
1/4 x 1/4 = 1/46 = 1/4,096). Remember that for
these calculations, we must make the assumption E = genus, Escherichia
that the nucleotides are distributed randomly. We co = species, coli
know that DNA molecules have very specific R = strain RY13
sequences of nucleotides, thus a given restriction I = first endonuclease isolated from this
site can occur either more or less often than the bacterium
above calculation predicts. Table 1. Selected restriction enzymes.
Enzyme Restriction Site End Products
B. Restriction Enzyme Nomenclature
BamHI GiGATCC Sticky
There are over 300 different restriction enzymes
that are now commercially available (see Table 1 AluI AGiCT Blunt
for examples). They are purified from a number EcoRI GiAATTC Sticky
of prokaryotic organisms and are named according BssHII GiCGCGC Sticky
to the following nomenclature: PstI CTGCAiG Sticky
HindIII AiAGCTT Sticky
1. The first letter is the first letter of the genus
name of the organism from which the enzyme
is isolated. C. Restriction Enzymes As Tools

2. The second and third letters are the initial
letters for the name of the species.
3. A fourth letter, if any, indicates a particular
strain of the organism.
4. Roman numerals indicate the sequence in
which different endonucleases were isolated
from a particular organism.
Restriction enzymes are frequently used to make
recombinant DNA for the purpose of cloning.
Plasmids can be engineered to contain specific
restriction enzyme recognition sites, and the same
restriction enzyme can be used to cleave both
the plasmid DNA and the DNA to be inserted
into the plasmid, creating DNA fragments with
complimentary sticky ends. DNA ligases can then
be used to join the different pieces of DNA together.
The resultant recombinant DNA molecules are
then introduced into host cells for replication or
protein expression.`

Restriction endonuclease digestion is also an

important step in DNA fingerprinting, a procedure
used in forensic investigations and paternity
inquiries to identify individuals from their DNA.
DNA fingerprinting is possible because all
individuals have natural variations in their DNA
sequences. In fact, it has been estimated that
nucleotide differences between individuals can be
detected approximately every 200 base pairs. These
genetic differences, or DNA polymorphisms,
create a genetic fingerprint that can be used to
specifically identify an individual from his or her
DNA. One way to produce a DNA fingerprint for
an individual is to perform a restriction fragment
length polymorphism (RFLP) analysis. RFLPs
are variations in the length of restriction fragments
produced from restriction enzyme digestion of
the DNA. The fragment lengths will vary between
individuals due to DNA polymorphisms, which
will effect the number and location of restriction-
enzyme recognition sites in the genome.

To produce a DNA fingerprint using RFLP

analysis, DNA is first isolated from a biological
sample, such as blood, hair, semen, or skin. Then
the DNA is subjected to a restriction-enzyme
digestion, which will chop up the DNA into
numerous different-sized fragments. The digested
DNA is then subjected to gel electrophoresis
to separate the fragments on the basis of size.
Although the DNA fragments can be visualized at
this step using ethidium bromide, the number of
DNA fragments on the gel would be too numerous
for the detection of a distinct banding pattern, and
would appear as a smear. In order to create a
distinctive banding pattern, the DNA fragments are
then probed using a short segment of radioactive
DNA that will bind to specific sequences within
the sample. Because only certain bands will be
labeled with this probe, the result will be a unique
barcode-like picture of an individual’s DNA.