Exercise 4: Introduction to Restriction Endonucleases
(see also pp. 108-113 in DNA Science)
Restriction Endonucleases
Bacterial restriction endonucleases were originally discovered as part of a defense
system against infection by bacterial viruses. These restriction endonucleases (also know as
‘restriction enzymes’ or ‘REs’) hydrolyze certain phosphodiester bonds within specific DNA
recognition sequences. This activity is useful in preventing infection because the bacteria
can partially hydrolyze the DNA of an infecting virus, if the virus has the sequence that is
recognized by the restriction enzyme. You can see, then, that having one restriction
endonuclease does not completely prevent a bacterium from infection by bacterial viruses
because each restriction endonuclease only cleaves the DNA of viral strain that contain the
specific DNA recognition sequence. To increase their resistance to infection, many bacteria
synthesize multiple restriction enzymes.
REs only bind to and act on double stranded DNA. This is because most REs are
active as symmetrical homodimeric proteins. Each monomeric subunit of the RE recognizes
one half of a DNA recognition sequence, binds to it, and cuts one strand of DNA. This means
that the dimeric enzyme usually recognizes a palindromic sequence and cuts both strands of
DNA symmetrically about the central point in the recognition sequence. While most
restriction endonucleases recognize palindromic sequence, there are exceptions.
Regardless of whether the recognition sequence is palindromic or not, the positions of
the cut sites on each strand are symmetrically located within that sequence. For REs with
recognition sequences that are an even number of base pairs, the endonuclease could cut in the
middle, producing two ‘ends’ that have no single stranded component. These ends are called
‘blunt ends’. Most enzymes, however, do not produce blunt ends but rather cut at sites other
than at the center of the recognition sequence. This results in one DNA strand being longer
than the other on each of the two ends produced. Hence each end of the double stranded DNA
has a single stranded component called an ‘overhang’. These ends are also known as ‘sticky’
or ‘cohesive’ because they have complementary overhangs and are able to anneal to one
another. The DNA recognition site and sites of cleavage for the enzyme EcoR I are indicated
below:
▼
5'…G A A T T C…3'
3'…C T T A A▲G…5'
Because each restriction enzyme binds to DNA only at a specific recognition sequence,
we can take advantage of this activity and employ it in recombinant DNA technology. If we
know the sequence of a fragment of DNA, and if that fragment has a requisite recognition
sequence, we can use the specific enzyme that recognized that sequence to cut the DNA at a
desired site or sites. In this way, a large piece of DNA can be digested into smaller, more
manageable fragments. REs that recognize a four base pair sequence would be expected to
have restriction sites that occur in DNA on the average of once every 4x4x4x4=256 base pairs,
and so produce DNA fragments with a rather small average size. If six nucleotides are
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specifically recognized, then sites occur once every 4096 base pairs (=4x4x4x4x4x4) on
average. This is a reasonably ‘workable’ size for a DNA fragment; as a result, ‘six-cutters’
(restriction enzymes that recognize a 6-nucleotide sequence) are the most commonly used
enzymes in molecular biology.
Each restriction enzyme requires specific reaction conditions for optimal activity.
While nearly all operate optimally at 37º C, some very commonly used exceptions function at
25ºC while others cut best at 55º C. In addition to temperature, optimal buffer (pH) conditions
also vary between restriction enzymes. In some cases, an enzyme’s activity is very dependent
upon specific buffer conditions while in other cases an enzyme is active in a broader spectrum
of conditions. In practice, the specificity of these reaction conditions often creates a problem,
for example, when doing a ‘double digest’ (cutting a fragment of DNA with two different
restriction enzymes).
REs are named for the organism from which they are first isolated and characterized.
The first three letters of the enzyme name come from the name of the bacterial species, and is
optionally followed by a single letter or an Arabic number designating the strain. This is
followed by a Roman numeral indicating order in which this enzyme was isolated from that
organism relative to other REs from the same organism. The name part is usually pronounced
rather than spelled out, but the Roman numeral is always pronounced by the cardinal number
(one, two, three, etc.). For example, EcoR I (pronounced: E- coh-are-won) is derived from E.
coli strain R, and it was the first enzyme isolated from this strain; Bgl II (bagel-too) was the
second restriction endonuclease isolated from Bacillus globigii.
You will now use restriction endonucleases to digest DNAs of differing sequences,
thereby creating restriction fragment length polymorphisms (RFLPs). Polymorphisms are
inherited differences in DNA sequence within a population of individuals. By running the
digested DNAs on an agarose gel, we will be able to see the number and lengths of restriction
fragments that are in each DNA sample.
The next page lists several Web sites that provide nice overviews of DNA forensics
and/or DNA gel electrophoresis. Following page 10 of this section is a print out of the first
link on the list from the Department of Energy’s Human Genome Project Information Web
site, which describes forensic DNA typing. You will note that DNA typing by restriction
endonuclease digestion is no longer the method of choice for most forensic investigations, due
to the need for larger quantities of DNA and a more laborious procedure. However, we
selected this method for introducing forensic typing since it is also an effective way to teach
about the function and importance of restriction endonucleases.
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Web Sites with Overviews of DNA Forensics and Gel Electrophoresis
DNA Forensics
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/forensics.shtml
DNA Profiling from Wikipedia
http://en.wikipedia.org/wiki/Genetic_fingerprinting
Basics of DNA Fingerprinting
http://protist.biology.washington.edu/fingerprint/dnaintro.html
VNTRs
http://protist.biology.washington.edu/fingerprint/vntrs.html
DNA fingerprinting enters society
http://genome.wellcome.ac.uk/doc%5Fwtd020878.html
Discovering DNA fingerprinting
http://genome.wellcome.ac.uk/doc%5Fwtd020877.html
NOVA Online – Create a DNA Fingerprint
http://www.pbs.org/wgbh/nova/sheppard/analyze.html
DNA Detective
http://www.dnalc.org/ddnalc/resources/shockwave/dnadetective.html
Virtual DNA Fingerprinting Lab from the Partnership for Biotechnology and Genomics
Education
http://ppge.ucdavis.edu/index.php?option=com_content&view=article&id=71&Itemid=137
Virtual Gel Electrophoresis Lab from the Genetic Science Learning Center
http://learn.genetics.utah.edu/units/biotech/gel/
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Exercise 4: Introduction to Forensic DNA Profiling
DNA in the Courtroom
Name___________________________ Date____________________________
BACKGROUND:
The experiments that generate the DNA profile are a bit more complex than portrayed
on CSI—but then again, they need to do everything in less than an hour! Many of them
involve the use of restriction enzymes and agarose gel electrophoresis. In these experiments,
you will simulate some of the steps involved in obtaining the type of evidence that appears in
the courtroom in cases utilizing DNA profiling. In this laboratory activity, you will be
investigating a murder case.
Each lab team will be provided with six different DNA samples. One was isolated
from the crime scene ("CS"), and does not belong to either of the victims. The other five DNA
samples have been isolated from five different individuals, any of whom may have been
involved in the murders ("S1," "S2," "S3," "S4," “S5”--suspects 1, 2, 3, 4, and 5). You will
digest each of these DNA samples with two restriction enzymes and separate the fragments on
an agarose gel. After staining, you will observe the distribution of bands on the gel. Then, you
will use the results to decide whether there is enough evidence to determine if any of the
suspects committed the murder. Remember, though, that this is a simulation. In reality, when
you cut human DNA with a restriction enzyme, millions of bands will be generated. Special
techniques allow the molecular biologist to analyze only specific regions of particular
chromosomes, rather than the entire genome in any one test. In addition, DNA profiling is
frequently done multiple times, using different combinations of restriction enzymes, on
different chromosomes in an effort to obtain multiple samples for analysis from any one
subject.
*************
Read the "news story" on page 6 to set the scene….
Abbreviations
CS
S1
S2
S3
S4
S5
=
=
=
=
=
=
DNA obtained at the crime scene
Suspect 1--John Gardener
Suspect 2--Dick Sateen
Suspect 3--Matthew Marshall
Suspect 4--Gail Greeley
Suspect 5—Richard McIntyre
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2 Popular Professors Killed at Liggett
St. Marie Shores, ME, Jan. 28--The safe, close-knit feel of Liggett College was shattered
today by the apparent murders of two popular, longtime professors, a couple known for
opening their home and hearts to others.
Gudrin and David Marshall had welcomed so many guests into their home "they practically
seemed to run a hotel," said colleague Brian Denton.
Police said the deaths were considered a double homicide.
The couple's latest guest had arrived at their home Saturday evening and found the door
unlocked, said neighbor and friend Arlene McIntyre.
"She went in and called out; there was no answer," McIntyre said in an interview. "She turned
and saw Gudrin on the floor, with blood all around her."
The guest, identified by others as Liggett biochemistry instructor Gail Greeley, rushed to the
McIntyre home to call the police. McIntyre said her husband, Richard, a doctor, then went to
the Marshall home.
"He saw enough to know for certain they were both dead and had been dead for a number of
hours," Arlene McIntyre said.
Gudrin Marshall, 53, was a math professor and chair of the Liggett Mathematics Department.
Her 51-year-old husband taught English Literature. They had been instructors at Liggett for at
least 15 years, said Martha Byron, dean of faculty for arts and sciences. The couple had two
adult sons: Victor, 25, who lives in San Francisco, and Matthew, 23, of Hanover, NH.
The younger son, Matthew, was visiting St. Marie Shores this weekend, but had been staying
with some friends. He and his parents had recently quarreled over money matters. He had not
commented on his parents' apparent murder, but has provided a DNA sample to police.
Police have also asked the Marshall's Saturday evening dinner guest, Gail Greeley, to provide
them with a DNA sample. They say, however, that she is not their main suspect at this time.
Two other persons have been asked for DNA samples. They include 16-year-old John
Gardener and 17-year-old Dick Sateen from neighboring Walled Lake, ME. It is unclear what
relationship these two young men had with the murdered professors. Nevertheless, their email addresses were found as recent entries in Gudrin and David Marshall's internet address
book. It appears as though the murder victims and the teens may have participated in the same
winter biathlon contest recently.
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Exercise 4: Analysis of DNA by Agarose Gel Electrophoresis
(see also pp. 113-115 in DNA Science)
Electrophoresis is the method used to separate charged molecules according to their
movement under the influence of an applied electric field. Electrophoresis of proteins has
been a standard technique since the 1930's, but the widespread application of electrophoresis
to nucleic acids has occurred only during the last 25 years. As is true of much current research
on nucleic acids, the majority of electrophoretic separations of DNA depend upon the ability
of particular enzymes -- referred to as restriction endonucleases -- to cut DNA molecules
into smaller pieces at precisely defined sites. You will have the opportunity to do a restriction
digest later in the workshop.
Nucleic acids are ideal candidates for electrophoresis because of their negative charge
and, with few exceptions, their three-dimensional shape. All that is needed is some inert
matrix through which the DNA or RNA can be driven by the electric field. One such matrix is
made from agarose. Agarose is a highly purified and uncharged polysaccharide obtained from
kelp; it is a component of the more crude agar used in making nutritional media for bacteria,
yeast, or humans (no kidding; agar is used to artificially thicken the heads of many
commercial beers). The porosity of an agarose gel is inversely related to the concentration of
agarose in the gel -- the lower the concentration of agarose, the more porous the gel. By
choosing the appropriate concentration of agarose, one can obtain a gel suitable for separating
either large DNA molecules, such as those of medium-sized viruses, or at the other extreme,
small DNA fragments consisting of only 50 base pairs. The mobility of a nucleic acid
molecule through gel pores is inversely related to the log of its molecular weight (or, as
another measure, its length in base pairs).
DNA by itself cannot be seen in a gel, and must be treated with something to be
detected. This is most commonly done by adding ethidium bromide to the agarose gel
mixture, which binds to the DNA. This highly fluorescent nucleic acid-ethidium bromide
complex can then be observed under UV light. Not surprisingly, a dye that binds to DNA is
highly mutagenic. Thus, work carefully with solutions of ethidium bromide, since it is a
known carcinogen.
PART I-- Pouring a 1% Agarose Gel for the DNA Fingerprinting Lab
1. Seal the ends of the gel-casting tray. Newer gel rigs, such as the GeneMates we use at
Amherst, use rubber gaskets in the gel tray itself to seal the end of the gel tray against the
gel box. Once completed, set the tray/gel box in a place where it will not be disturbed for
the next hour or so.
2. Position the well former (with the comb with 8 wells facing downward) in the notches at
one end of the tray (note that the teeth are raised ~1 mm above the Plexiglas surface). Ask
your instructor for assistance and observe the demonstration: if the well former contacts the
bottom of the tray, then your DNA samples will run under the gel, not through it; at the
other extreme, if the well former is too shallow, you may not be able to load enough DNA
samples to observe later.
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3. Prepare the gel solution by mixing 50 ml of 1X TAE with 0.5 g of agarose in a 125 ml
flask. Heat the mixture in a microwave oven to dissolve the agarose. Swirl the flasks
occasionally (caution: they get hot; handle with gloves, hot hands or paper towels), and
bring just to a boil, but be careful not to let the mixture boil over. Allow the mixture to
heat for ~60 seconds, then remove and swirl the mixture to help dissolve the agarose, and
then heat the mixture for another 40-60 seconds. Afterward, remove the mixture from the
microwave and let the agarose cool until the flask is cool enough to touch and hold without
needing paper towels or gloves.
4. Carefully pour the molten agarose into the casting tray; the agarose should cover the bottom
one-half to three-quarters of the comb. This should use all of your molten agarose.
5. The gel will become cloudy as it solidifies (15-20 minutes). It is essential that the gel box
not be moved during this time. BE PATIENT: it's better to wait an additional 5 minutes for
the gel to set than to have your well former plop into the gel and then have to start all over.
6. When the gel is completely set, CAREFULLY pull up the gel tray from the casting box.
***Overnight Storage: put gel tray (with comb in place) into a Ziploc bag in refrigerator***
7. Add enough 1X TAE to the gel box to cover the middle raised platform, and place the gel
tray on the platform, parallel to the long axis of the gel box. Fill the box with TAE buffer
(~270 ml), to a level that just covers the entire surface of the gel.
8. GENTLY tease out the comb, by wobbling it a little while pulling up on it (be careful not to
tear the wells).
9. Make sure that the gel is completely submerged in the 1X TAE before proceeding.
PART II---Loading the Gel and Electrophoresis
1. You will add 5X loading dye (LD) to each of the DNA samples before loading them into
the gel. The LD contains glycerol or sucrose, to make the samples dense and therefore
easier to load into the wells, and a tracking dye that will let you monitor the progress of
your samples through the gel. The tracking dye, like DNA, is negatively charged and will
run ahead of the larger DNA molecules.
2. Add 5 µl of 5X loading dye to each reaction tube from Day 1. Mix well by either tapping
the tube on the lab bench or by flicking the end of it with your finger tip. Do a quick spin in
the microfuge to bring all the solution back to the bottom of the tube.
3. Use a P20 micropipettor at the proper setting to transfer the contents of each reaction tube
to the well in the gel as in step #7 on p. 21 of the Bio-Rad Manual’s Quick Guide. Use
a clean tip for each reaction tube. BE VERY CAREFUL--DON’T PUSH THE TIP
THROUGH THE BOTTOM OF THE WELL.
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4. Close the top of the gel box and connect the electrical leads to their correct electrodes: Red
to Red and Black to Black. Check carefully that the high voltage power supply is OFF.
Check again. Connect the leads from the gel box to the power supply, making sure that the
DNA will migrate across the length of the gel towards the anode (+).
5. Turn on the power supply and gradually increase the voltage to 100 volts. Use caution
when working with high voltage and ask for assistance! Observe the gel after 5 to 10
minutes: you should be able to see that your samples are moving in the proper direction
from the migration of the dye. Allow the electrophoresis to proceed for ~1 hour, checking
the run periodically for the position of the sample dye. When finished, the dye should be
within 1.5 cm of the end of the gel, but ask a lab instructor to help when you think it might
be finished.
6. When gel is finished running, dial the voltage down to zero, turn the high voltage power
supply to OFF, and unplug the unit. Now you are ready to disconnect the electrodes and
open up the gel box.
PART III---Staining your gel
1. Before you remove the gel tray from the box, put on gloves.
2. Follow the Overnight staining directions on p. 22 of Bio-Rad’s Quick Guide to stain the
gel overnight with 1X Fast Blast (1:500 dilution). Avoid touching the top of the gel. Two
groups should coordinate with one another, as two gels can stain together in one staining
tray.
3. Place the gel trays on a rotary shaker for overnight staining.
PART IV---Visualizing your gel bands
1. Pour off the Fast Blast into a waste container and rinse the gel(s) with 60 ml of water.
Rinse gels again, if necessary.
2. Place the gel(s) on the light box and photograph the gel. Note: two mini gels will fit
under the camera hood, so if you double up with another group, the photo can be cut
in half (saving photo paper!).
3. Place the gel on a piece of gel support film and allow gel to dry for 2-3 days.
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