Genetic Engineering

PRACTICAL Restriction Enzyme Digestion

Objectives
1. To cleave DNA using Type II restriction enzyme.
2. To familiarize student on how to do aseptic lab work.

Introduction
Type II restriction enzymes are the class usually used to cleave DNA. This is because cleavage
with these enzymes occurs at specific sites within or adjacent to the enzymes’ recognition
sites.

Materials and Methodology

The following was added into an eppendorf tube: DNA in TE (1µg), 10 X restriction enzyme
buffers (2µl), restriction enzyme (0.5µl), and water to make up 20µl (which is 17.5 µl).

The digestions are routinely done at 37°C unless lower or higher temperatures are required
for optimal digestion. RE buffers are normally supplied together with the enzymes.
Normally, 1 unit of enzymes is needed to digest 1µg of DNA at the appropriate temperature
in 1 hour.

Then 15µl of genomic solution after incubation with RE was loaded into the well in 1.2%
agarose gel. The gel was run at 75V at 5 mins and 70V at 40 mins. The agarose gel was
stained in the ethidium bromide and visualized under UV light.

Result

M: DNA molecular weight marker

U: uncut plasmid DNA
E: Digested with EcoRI

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Question
1. What is RE? Give types of RE.
RE is an enzyme, specifically an endode-oxyribonuclease, which recognizes a short
specific sequence within a deoxyribonucleic acid (DNA) molecule and then catalyzes
double-strand cleavage of that molecule. Restriction enzymes have been found only in
bacteria, where they serve to protect the bacterium from the deleterious effects of
foreign DNA. There are three known types of restriction enzymes:
• Type I enzymes recognize a specific sequence on DNA, but cleave the DNA chain at
random locations with respect to this sequence. They have an absolute requirement
for the cofactors adenosine triphosphate (ATP) and S-adenosylmethionine. Because
of the random nature of the cleavage, the products are a heterogeneous array of
DNA fragments.
• Type II enzymes also recognize a specific nucleotide sequence but they do not
require cofactors and they cleave specifically within or close to the recognition
sequence, thus generating a specific set of fragments. It is this exquisite specificity
which has made these enzymes of great importance in DNA research, especially in
the production of recombinant DNAs.
• Type III enzymes have properties intermediate between those of the type I and type
II enzymes. They recognize a specific sequence and cleave specifically a short
distance away from the recognition sequence. They have an absolute requirement
for the ATP cofactor, but they do not hydrolyze it.

2. Why we cut the genomic or DNA with RE? Explain.

Restriction enzymes are able to "scan" the length of a DNA molecule. It is looking for a
particular pattern of nucleotides, the enzyme's recognition sequence. Once it encounters
its specific recognition sequence, generally 4 to 6 nucleotides long, the enzyme will bond
to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones
of the double helix. Once the cuts have been made, the DNA molecule will break into
fragments. A restriction enzyme always cuts DNA at its recognition sequence, regardless
of whether the DNA is from a virus, a bacterium, a plant or an animal.

3. Give the application of RE in molecular technique.

• They are used to assist insertion of genes into plasmid vectors during gene cloning
and protein expression experiments. For optimal use, plasmids that are commonly
used for gene cloning are modified to include a short linker sequence (called the
multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This
allows flexibility when inserting gene fragments into the plasmid vector; restriction
sites contained naturally within genes influence the choice of endonuclease for
digesting the DNA since it is necessary to avoid restriction of wanted DNA while
intentionally cutting the ends of the DNA. To clone a gene fragment into a vector,
both plasmid DNA and gene insert are typically cut with the same restriction
enzymes, and then glued together with the assistance of an enzyme known as a DNA
ligase.
• They can be used to distinguish gene alleles by specifically recognizing single base
changes in DNA known as single nucleotide polymorphisms (SNPs). This is only
possible if a SNP alters the restriction site present in the allele. In this method, the
restriction enzyme can be used to genotype a DNA sample without the need for
expensive gene sequencing.
• Restriction enzymes are also used to digest genomic DNA for gene analysis by
Southern Blot. This technique allows researchers to identify how many copies of a
gene are present in the genome of one individual, or how many gene mutations

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(polymorphisms) have occurred within a population. The latter example is called

Restriction Fragment Length Polymorphism (RFLP).

Conclusion
Through this experiment, we become familiar with the aseptic technique to cleave the DNA
using Type II restriction enzyme.

References
"restriction enzyme." McGraw-Hill Encyclopedia of Science and Technology. The McGraw-
Hill Companies, Inc., 2005. Answers.com (250908)
www.answers.com/topic/restriction-enzyme

http://en.wikipedia.org/wiki/Restriction_enzyme (250908)