BZE F2020 Lab 7

Restriction Enzymes & Gel Electrophoresis

READ pages 437 – 440 (up until “Amplifying DNA”) + figures 20.6 & 20.7 page 439

AIMS
When you have completed this lab, you will be able to:
A. Define restriction enzymes and their role in bacteria;
B. Understand the role of restriction enzymes in biotechnologies;
C. Learn to separate DNA on an agarose gel using electrophoresis;
D. Understand how to use a restriction digestion map to identify a sample DNA.

INTRODUCTION
A) Restriction Enzymes

Bacteriophage (also called phages) are a type of virus that infect bacteria by injecting nucleic acids
(DNA or RNA) into the bacterial cell. In so doing, the virus “hijacks” the bacterial DNA replication &
transcription/translation machinery (enzymes and non-enzyme proteins) to make new virus instead of
new bacteria. In the lytic cycle (lysis means breaking of the cell) illustrated in figure 1, the bacterial
cell, after some time, bursts, releasing hundreds of new bacteriophages that continue the infection
process. See page 419 & figure 19.6 page 419 in your textbook.

Figure 1: lytic cycle in phage infection of a bacterial cell

Bacteria are not defenseless against these infections. First, natural selection favors bacterial mutants
with membrane receptors that do not recognize, & therefore do not bind, different types of phages.
Second, when phage DNA successfully enters a bacterium, the viral DNA is often identified as foreign
and hydrolyzed by cellular enzymes called restriction enzymes, specifically restriction
endonucleases. Restriction endonucleases are members of a larger group of enzymes called
nucleases that function in breaking the phosphodiester bonds that link adjacent nucleotide
monomers in DNA. Endonucleases cleave DNA at internal positions, whereas exonucleases
progressively digest from the ends of DNA molecules.
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Figure 2: restriction enzyme cleavage of DNA phosphate-sugar backbone

Restriction endonucleases act as chemical “scissors”, fragmenting the viral DNA as soon as it enters
the bacterial cell. Restriction enzymes scan the length of the viral DNA & recognize specific nucleotide
sequences, called recognition or restriction sites, in the double-stranded DNA. Once a recognition
site is located, the restriction enzymes bind to the DNA & cleave each strand of the double helix at a
specific place within the recognition site. The restriction enzymes will do this along the full length of
the viral DNA molecule, which will then be broken into fragments – stopping the infection in its tracks.
The size of the fragments is measured in number of base pairs (bp) or kilobases (1000 base pairs -
kbp).

Figure 3: restriction enzyme digest of viral DNA & its degradation into smaller fragments

B) Recognition Sites and Palindromes

Recognition sites for restriction enzymes occur randomly along DNA sequences & generally consist
of short, 4 – 6 bp in length, symmetrical sequences or palindromes. Palindromes are groups of letters
that read the same in both forward & backward orientation. In the case of DNA, the nitrogenous bases
of the nucleotides are the letters which are repeated in opposing orientations on both strands of the
DNA double helix. For example, the 5’ to 3’ strand of the DNA double helix may have the sequence
GAATTC. The complimentary bases on the 3” to 5” strand will therefore be CTTAAG – which is
GAATTC backwards.

Many restriction endonucleases recognize these types of palindromes, attach to the viral DNA at these
specific recognition sites & cut the DNA strand between two of the bases. For instance, the restriction
endonuclease called EcoRV from the bacterium E. coli recognizes & cuts the sequence 5’ GATATC
3’ in double-stranded DNA at the AT & AT junctions. This allows each half of the restriction enzyme
scissors to recognize the same sequence on both DNA strands.

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When EcoRV cuts the double-stranded DNA it leaves

fragments with blunt ends. However, many
restriction endonucleases cleave their palindromic
recognition sequences asymmetrically, leaving a
single-stranded overhang at each side of the cut,
called a sticky end. At any given enzyme recognition
cleavage site, these overhangs have unique and
complementary sequences.

Figure 4: sticky end products of restriction enzyme digest

For example, the restriction enzyme HindIII from the bacterium H. influenzae recognizes the
palindrome 5’ AAGCTT 3’ & cleaves the DNA helix between the first two A of the recognition site.

The ragged or "sticky" ends that remain after restriction enzyme cleavage are a key aspect of
recombinant DNA technology, covered below.

C) Methylation

Bacterial restriction endonucleases target viral DNA, not the bacterial cells' own DNA, because
bacteria modify their own DNA, using enzymes known as methylases that add methyl (CH3) groups
to some of the nucleotides in the bacterial DNA.

Figure 5: example of methylation of the nitrogenous base of a nucleotide

When nucleotides within a restriction endonuclease's recognition sequence have been methylated,
the endonuclease cannot bind to that sequence. Consequently, the bacterial DNA is protected from
being degraded at that site. Viral DNA, on the other hand, has not been methylated & therefore is not
protected from enzymatic cleavage.

Figure 5: example of methylation of the nitrogenous base of a nucleotide

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D) The Use of Restriction Enzymes in Biotechnologies

I. DNA Fingerprinting

DNA fingerprinting, also called DNA profiling or DNA barcoding, is the process of determining
an organism’s or a specie’s DNA characteristics. DNA fingerprinting is often used in scientific
fields such as medicine, microbiology, zoology, botany & agriculture. It’s also used as a forensic
technique in criminal investigations, comparing suspects' profiles to DNA evidence, in parentage
testing & in genealogical research. Several methods of DNA fingerprinting use restriction
enzymes.

II. Recombinant DNA

As indicated above, the sticky ends that remain after cleavage by certain restriction enzymes
are key to producing recombinant DNA. Cleavage of DNA from any organism with these specific
restriction enzymes always generates the same complementary ends on either side of the cut. In
the presence of specific DNA repair enzymes, the DNA fragments will reanneal themselves to
other fragments with sticky ends that are complimentary to their own end sequence. It doesn’t
matter if the fragment that matches the cut end comes from the same organism or from a different
one . For example, a human DNA restriction fragment can be fused with the sticky end of bacterial
DNA molecule that was cut with the same restriction enzyme. DNA synthesis & repair enzymes
catalyze the formation of phosphodiester bonds across the breaks & the backbone of the resulting
recombinant DNA from a human & a bacterial cell is indistinguishable from the original uncut
DNA. There is no limit to the design of such DNA rearrangements, as long as there are appropriate
restriction sites in the DNA sequences to be restructured.

This ability of DNA to be cut & then repair itself has been utilized by scientists to introduce foreign
DNA into several different organisms, often resulting in those recombinant organisms exhibiting
new characteristics or functions. Examples include a change in the nutritional quality of a plant,
or the production of human proteins, such as insulin, by bacterial cells.

E) Gel Electrophoresis

The size of a given fragment will depend upon the distance between two recognition sites along
the DNA molecule. The number of fragments obtained using a restriction enzyme will depend upon
how many restriction sites are present in the DNA. For example, a piece of linear DNA with 2
recognition sites would produce 3 smaller fragments upon hydrolysis by the enzyme.

The fragments produced by treatment of a DNA sample with restriction endonucleases are separated
by means of electrophoresis in a gel . The gel is made of a polysaccharide derived from agar, called
agarose. Agarose gel electrophoresis is a widely used analytical tool that separates molecules not
only on the basis of differences in charge, but also in size and shape. It is particularly useful for
separating biomolecules such as DNA, RNA & proteins. It is a type of electrophoresis in which the
gel, in addition to carrying the current, also acts as a molecular sieve. Small molecules placed in the
gel move easily through the small pores in the gel lattice, while large molecules have more difficulty.
As a result, for molecules of similar shape and charge, the smaller the molecule, the further it will
move in agarose gel.

In the separation of DNA fragments, size is the most important factor. Charge & shape are not
important because DNA fragments are all negatively charged (the movement of the fragments will
always be towards the positive electrode) & all are about the same shape. Therefore, after
electrophoresis, all the fragments in a given band will be about the same size.

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Figure 6 below provides an example of the analysis of a gene using restriction enzyme digest & gel
electrophoresis. Notice that there are 4 sites for the restriction enzyme DdeI in the wild type ß-globin
gene, producing 3 fragments of different sizes. In the mutant form of the gene, there are only 3 sites,
producing 2 fragments of different sizes.

Figure 6: analysis of wild type & mutated ß-globin gene using restriction enzyme digest & gel electrophoresis

Figures 7 - 9 provide an example of DNA analysis using restriction enzyme digest and gel
electrophoresis in parentage testing. A child's DNA is a composite of half of each parent's DNA (rare
exceptions, called chimeras, notwithstanding). Therefore, the DNA fingerprints of a mother and child
will have some bands in common, & the bands in the child's fingerprint that are not present in the
mother's must have been contributed by the father. In other words, each band in the child's fingerprint
should be found in either the mother's or the father's fingerprint (unless a crossing over event has
occurred within that DNA sequence).

Figures 7 – 9: parentage testing using restriction enzyme digest & gel electrophoresis

F) Restriction Digest & Gel Electrophoretic Analysis of Recombinant Plasmid pGLO

In this lab we will be using gel electrophoresis to study the restriction fragments obtained from a 5.4kbp
in length recombinant plasmid DNA, called pGLO plasmid.

Bacteria have one large circular chromosome & one or more plasmids. A plasmid is a small
circular extrachromosomal (not part of any chromosome) DNA that contains genes for traits that may
help the bacteria survive under specific conditions. Plasmids are also found in yeast.

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Plasmids replicate independently of the chromosomes

Figure 10: illustrative representation of the one circular bacterial chromosome & several plasmids

In nature, bacteria can transfer plasmids back and forth between individual cells, allowing them to
share the beneficial genes present on the plasmids.

pGLO is a bacterial plasmid in which genes from other organisms have been inserted using
recombinant DNA techniques.

PROCEDURE
See the following YouTube video (4 minutes) before continuing…
https://www.youtube.com/watch?v=vq759wKCCUQ

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Part 1: Preparation of the enzyme digests and the agarose gel

Three separate digests were prepared for you:

1. EcoRV pGLO digest
2. HindIII pGLO digest
3. EcoRV + HindIII pGLO digest

Each digest was prepared by adding water, followed by pGLO DNA, a buffer solution and finally the
specific restriction enzyme (EcoRV, HindIII or both). Once mixed together, the three microtubes, were
placed on ice.

The microtubes were then placed in a water bath at 37°C for 15 minutes. Restriction enzymes work
best at 37°C since they were isolated from bacteria that live inside warm-blooded animals. Once the
incubation was done, the samples were placed back on ice.

An agarose gel was prepared by mixing TAE buffer with agarose powder and heating it up to dissolve
the powder. SYBR green dye was added to the mixture and gently mixed into the solution.

Note: A very important step in preparing a gel is the addition of a comb at the 0 cm mark. This comb
will ensure that once the gel cools down (and becomes solid), the comb will have left wells in the shape
of the gel which will be used to load our samples.

The gel was removed from its casting tray and inserted in the electrophoresis chamber and immersed
in more buffer. The cathode was placed at the negative end (at the 0 cm mark, where the wells are
located in the gel) and the anode at the opposite end.

Part 2: Preparation of the samples and the ladder for loading

DNA ladders are standards used to visualize the size of each restriction fragment of a sample by
comparing it to the known size of the fragments (like “rungs”) on the DNA ladder. It comes already
mixed and prepared in many cases.

Part 3: Loading the samples and running the gel electrophoresis

Gels are read from left to right. Your gel was loaded as such:

Lane 1 2 3 4
Sample Ladder (L) HindIII pGLO (H) EcoRV pGLO (E) EcoRV + HindIII pGLO (EH)

Loading dye was added to each sample to help you visualize the movement progression of the
samples through the agarose gel. Each sample was pipetted in a separate well in the agarose gel.

The gel underwent electrophoresis for 30-45 minutes at 100 V.

Part 4: Visualizing the agarose gel

The agarose gel was visualized using a UV trans-illuminator

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LAB REPORT
Names ______________________________________________ ID ______________

1. Each of the restriction enzymes below only recognizes a specific palindrome and cuts
DNA only at that specific sequence. Complete the table below.
Restriction enzyme Recognition sequence
5’ G A A T T C 3’
Eco RI
3’ 5’
5’ A A G C T T 3’
Hind III
3’ 5’
5’ C T G C A G 3’
Pst I
3’ 5’
2. A linear DNA molecule is cut with Eco RI restriction endonuclease, generating 4
fragments. Eco RI cuts this DNA how many times? _________ times

3. Agarose gel electrophoresis is a procedure used to separate DNA fragments. Based on

what characteristic do the fragments separate?

4. A piece of DNA is cut into 4 fragments as shown in the diagram below.

a. A solution containing the four fragments is placed in a well in an agarose gel. In the
diagram below, draw how you think the fragments might be separated. Label each
fragment with its corresponding letter, A, B, C, or D.

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5. You decide to digest a 14,000 bp plasmid that has only 1 restriction site.
a. Draw the approximate band(s) that you would expect to see.

b. Explain your result (how many bands? size?) as shown on the gel above.

6. Below is a plasmid with restriction sites for Bam HI & Eco RI. Several restriction digests
were done using several combinations of these two enzymes. Use the figure to answer
questions 6a. to 6c.

a. Which lane shows a digest with Bam HI only? Lane _____

b. Which lane shows a digest with Eco RI only? Lane _____
c. Which lane shows the fragments produced when the plasmid was incubated with
both Eco RI & BamH1? Lane _____

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7. Explain why restriction endonucleases do not cleave a bacterial cell's own DNA as well as
that of the viruses that are infecting it.

8. From the gel fingerprinting results seen below, determine which pair represents the actual
parent of the child.

a. The parent pair is _____.

b. Show your reasoning below OR indicate it on the gel above with tracings.

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9. The figure below shows you results from the experiment you should have conducted in
the lab. The image has been taken from a team registered in BZE in a previous year.

From the fragment pattern shown above, reconstruct the pGLO recombinant plasmid by
determining below the relative positions of the recognition sites for the two enzymes
used. Remember that the pGLO plasmid is ~5.4Kbp (5,400 base-pairs) in total.

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