Techniques: Restriction Nuclease Mapping
and DNA Electrophoresis
Background
Restriction mapping is a widely used method in recombinant DNA research. It is an
important adjunct to DNA sequencing methods and even by itself, reveals a great deal
about the molecular architecture of a DNA molecule. The general procedure involves
treating a sample of DNA with restriction enzymes, which cleave the molecule at specific
sites. The method for locating the cleavage sites along the DNA molecule is referred to as
restriction mapping. Restriction maps were first used to study the relatively simple
genomes of plasmids, viruses, and bacteria.
Restriction mapping, like many other methods used in molecular genetics research,
requires the use of a special class of enzymes, the restriction endonucleases, referred to
simply as restriction enzymes. The term endonuclease indicates that these enzymes
cleave internal phosphodiester bonds of polynucleotide chains. Each restriction enzyme
recognizes a specific base sequence and cleaves the DNA molecule at every such
recognition sequence, or recognition site, producing “blunt” or “sticky” ended fragments.
Thus, when a DNA molecule is treated with a particular restriction enzyme, the DNA is
cut into a characteristic number of fragments, termed restriction fragments. Finally, most
restriction endonucleases are sensitive to extreme heat and can be inactivated. This is a
very convenient method for stopping a restriction endonuclease reaction (incubation at
65°C for 20 minutes) which performs optimally at 37°C. For restriction mapping, the
DNA is treated with restriction enzymes individually and in various combinations.
The second step in restriction mapping entails separating the DNA fragments with the
technique of gel electrophoresis. The enzymatic digests, each containing a number of
restriction fragments, are placed in sample wells near one end of an agarose gel. The
DNA fragments, which are negatively charged (because of their phosphate groups),
migrate toward the anodic (positive) end of the gel at a rate that is a function of molecular
weight or length. The distances migrated by the DNA fragments can be used to estimate
their lengths.
Once the lengths of the restriction fragments produced by two or more restriction
enzymes are known, it is possible to locate the cleavage sites of the enzymes, as the
following example illustrates. Consider a DNA molecule containing 50,000 base pairs.
The length of a long polynucleotide chain is conveniently given in kilobases (kb),
corresponding to the number of kilobase pairs. Thus, the length of this DNA molecule
can be written as 50 kb. Now, consider the two restriction enzymes, RE1 and RE2. If, in
this DNA molecule, there is one recognition sequence for each of these restriction
enzymes, treatment with either enzyme would produce two restriction fragments.
Assume that the electropherogram obtained from treatment of this DNA with RE1 is
depicted in the figure below. In lane 1, there are two bands; the band closer to the top of
the gel (where the sample was originally loaded) contains fragments that are 35kb long,
whereas the band closer to the bottom of the gel contains fragments that are 15kb long. In
lane 2, we see the pattern of bands that results when the DNA molecule is treated with
RE2. Here, the two fragments produced are 40kb and 10kb.
We would like to know in which of the two restriction fragments produced by RE1 (35kb
or 15kb) is the recognition sequence for RE2 located. The answer is obtained by treating
the DNA with both enzymes simultaneously and then comparing the lengths of the
fragments in the double digest (lane 3) with the lengths of the fragments obtained after
treatment with RE1 alone (lane I). As can be seen in the figure, both of these lanes have a
35kb fragment, but the 15kb fragment is missing from lane 3, having been replaced by a
10kb fragment and a 5kb fragment. Clearly, then, the cleavage site for RE2 is located
within the 15kb fragment. A comparison of the fragments produced in the double digest
with the fragments produced using RE2 alone reveals that the cleavage site for RE1 lies
within the 40kb fragment.
The figure shows the restriction map with the cleavage sites of both restriction enzymes
indicated. In this simple example, the left and right ends of the DNA molecule (0 kb end
and 50 kb end, respectively) are not identified. If the cleavage site in relation to the ends
were known for one restriction enzyme, it would be known for the other restriction
enzyme when the restriction map was completed.
DNA Restriction Analysis of Phage  DNA
In this experiment, DNA from the bacteriophage lambda (48,502 base pairs in length) has
been cut with restriction enzymes. You will separate the resulting restriction fragments
using gel electrophoresis.
Three samples of lambda DNA have been incubated at 37°C, each with one of three
restriction endonucleases: BamHI, EcoRI or Hindlll. The DNA samples are then loaded
into wells of an agarose gel and electrophoresed. An electrical field applied across the gel
causes the DNA fragments in the samples to move from their origins (sample wells)
through the gel matrix toward the positive electrode. Smaller DNA fragments migrate
faster than larger ones, so restriction fragments of differing sizes become concentrated
into separate bands during electrophoresis. The characteristic number and pattern of
bands produced by each restriction enzyme are a “DNA fingerprint.” The restriction
patterns are made visible by staining with a compound that binds to DNA.
Purpose
The purpose of this exercise is to familiarize you with agarose gel electrophoresis and the
concept of restriction nuclease mapping.
Materials per team
P20 pipetman
Pipetman tips (yellow)
50 mL 1X TBE buffer
Digested DNA samples (EcoRI and HindIII) in loading dye and Sybr-Green Stain
Calculator
Two 100 mL beakers
Agarose powder
1X Gel Running Sample
Weighing boat
Digital balance
Hot plate
Gel cast tray with a comb
Masking tape
Electrophoresis apparatus
Metric Ruler
Power supply
Double Combed, Pre-cast Practice Gels (prepared by your instructor before class)
Procedure
1. Place a weighing boat on the scale. Observe the weight of the boat in the digital
display. With the boat on the scale, tare the balance to 0.0 g by depressing the ‘zero’
button. You are ready to weigh a sample now.
2. Dispense 1.2 g (gram) of agarose powder into the boat on the digital balance.
3. Mix the agarose with the 100 mL of TBE buffer in the 100 mL beaker and bring to a
‘simmer’ on the hot plate. You want to melt the agarose powder into solution; this
takes about 10 minutes on the hot plate. When the agarose powder is completely
dissolved (solution is miscible), take it off of the hot plate and let it cool on the
bench-top for about 15 minutes. Now we prepare the gel cast tray and agarose gel.
4. Tape both ends of the gel tray and affix the comb (the instructor will show you how).
Make sure the seals are firm or else the warm, liquefied agarose solution will leak
from the gel during pouring and subsequent molding. Pour approximately 40 mL of
warm (if you do not burn your hand holding the beaker, it is the perfect temperature)
liquid agarose into the gel cast tray. Let the agarose gel solidify (20 minutes), then
proceed to step 9. Now let’s practice loading a sample into a gel.
5. Pick up a pre-cast, instructor-prepared agarose gel. You should see indentations in
the gel caused by the comb; this is where you will load your sample.
6. Grab a P20 pipetman and set the dispensation volume to 10 L. Affix a yellow
colored pipetman tip to the pipetman dispenser.
7. Measure your sample from the microcentrifuge tube containing 1X gel running
sample. Dispense your sample into a well in the tray (the instructor will show you).
8. Each student takes turns loading a sample into the agarose gel. Now to the real thing!
9. Pick up the solidified agarose gel (in the casting tray) and gently peel off the masking
tape on both ends of the casting tray. Make sure the agarose gel stays in the tray!
Also, slowly and gently pull the comb up and out of the gel. You should see
indentations in the gel where you will load your sample.
10. Position the electrophoresis apparatus in front of you (lengthwise) with the 2 metal
poles furthest away from you (on the backside). Next, place the gel (still in the
casting tray) into the electrophoresis apparatus. Be certain to orient the gel
appropriately, with the wells closest to the (-; black color) left end of the apparatus
and furthest from the (+; red color) right end of the apparatus. Verify the proper
orientation with your instructor before you proceed.
11. Fill the electrophoresis apparatus with enough 1X TBE buffer to submerge the
agarose gel.
12. Grab a P20 pipetman and set the dispensation volume to 10 L. Affix a yellow
colored pipetman tip to the pipetman dispenser.
13. Measure your digested DNA sample from the microcentrifuge tube. Dispense your
sample into a well in the tray. Make note of what is loaded in each well of the gel.
14. Each student takes a turn loading a sample into the agarose gel.
15. When the samples are loaded, secure the lid on the electrophoresis apparatus,
connect the lid to the power supply, connect the power supply to an outlet, turn on
the power supply and run the samples on the medium setting. Take notice of the
bubbles coming up from the platinum wire on the far left. Current is being
conducted and gas is being generated.
16. Check back at the gel in 5 minutes. Where is the position of the dye? Bet it is out of
the well; your DNA sample is migrating in the gel. Its components are being
separated by their fragment size.
17. After the blue dye has migrated ¾ of the total distance of the gel, turn the power off.
18. Remove the gel and resolve the gel under UV light.
19. Look at the gel. Compare it to the reference data in the Appendix section of the
worksheet (this is enclosed at the end of this exercise – look on the next page).
WORKSHEET
Restriction Nuclease Mapping and DNA Electrophoresis
1. Why do you think restriction endonucleases are sensitive to extremes in heat?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
2. Do you think restriction endonucleases are also sensitive to extremes in cold? Why?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
3. What is the difference between a “blunt” or “sticky” ended fragment? Give one
example of each type of fragment and the endonuclease causing it.
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
4. What is agarose? Why does it separate DNA fragments according to length?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
5. An anion has a ___________ (negative or positive) charge. It will migrate to the
_____________ (cathode or anode)?
6. An ________________ (anion or cation) has a positive charge. So it will migrate to
the ___________ (anode or cathode), which is a ____________ (negatively or
positively) charged electrode.
7. DNA migrates to toward the ______________ (anode or cathode) because it is
___________________ (positively or negatively) charged.
8. DNA possesses phosphates which are ___________ (negatively or positively)
charged.
9. Describe three applications of agarose gel electrophoresis? (hint: Wikipedia)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
10. Do you think a gel containing 1.5% agarose would produce the same DNA fragment
resolution as a 0.8% agarose gel (ok, the answer is obviously no)? Why not?
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
11. DNA fragments of similar size will not always resolve on a gel. This is seen in the
EcoRI digest lane in the ideal gel attached in the Appendix (at the end of this
worksheet), where EcoRI fragments of 5804 bp and 5643 bp migrate as a single
heavy band (third band from the top). These are referred to as a doublet and can be
recognized because they are brighter and thicker than similarly sized singlets. What
could be done to resolve doublet fragments? (hint: think about question 10 and also
take a moment to look over the different types of agarose gels in the Field Guide
located in the Appendix – focus on the short vs. long run gels)
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
12. Now comes the really hard part. On the enclosed semi-log paper (appendix), you will
plot the electrophoretic mobilities of the standard DNA digest (HindIII) as a function
of their fragment lengths and determine the sizes of the unknown DNA fragments
(EcoRI and BamHI digests) in base-pairs. To help you, a genetic map of phage
lambda is enclosed in the Appendix.
Linear DNA fragments migrate at rates inversely proportional to the log10 of their
molecular weights. For simplicity’s sake, base-pair length is substituted for molecular
weight.
a. The matrix below gives the actual size in base pairs (Act. bp) of lambda DNA
fragments generated by a HindIII digest.
b. Using the ideal gel shown in the Appendix, carefully measure the distance (in
mm) each HindIII, EcoRI and BamHl fragment migrated from the origin.
Measure from the front edge of the well to the front edge of each band. Enter the
distances into the matrix above (enter under Dis.).
c. Match base-pair sizes of Hindlll fragments with bands that appear in the ideal
digest. Label each band with kilobase-pair (kbp) size. For example, 27,491 bp
equals 27.5 kbp.
d. Now it is time to prepare the Calibration Curve. Set up a semi-log graph paper
with the distance migrated as the x (arithmetic) axis and the log of base-pair
length as the y (logarithmic) axis. Then, plot the distance migrated versus the
base-pair length of each HindIII fragment.
e. Connect the data points with a best-fit line.
f. Locate on the x axis the distance migrated by the first EcoRI fragment. Using a
ruler, draw a vertical line from this point to its intersection with the best-fit data
line.
g. Now extend a horizontal line from this point to the y axis. This gives the base-pair
size of this EcoRI fragment.
h. Repeat steps f and g for each EcoRI and BamHl fragment. Enter the results in the
calculated base pairs (Cal. bp) columns for each digest.
i. Enter the actual base-pair size of EcoRI and BamHl fragments, as provided by
your instructor (refer to Appendix, under Restriction Digest Data), into the Act.
Bp column.
j. For which fragment sizes was your graph most accurate? For which fragment
sizes was it least accurate? What does this tell you about the resolving ability of
agarose gel electrophoresis?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
Appendix: Ideal Gel Data, Semi-Log Paper, Field Guide, Phage  Genetic Map
and Restriction Digest Answers
Genetic Map of the Phage Lambda () Genome and Restriction Digest Answers
Note: Lambda DNA can exist both as a circular molecule and as a linear molecule. At
each end of the linear molecule is a single-stranded sequence of 12 nucleotides, called a
COS site. The COS sites at each end of the linear form of lambda are complementary to
each other and thus, can base pair to form a circular molecule. These complementary
ends are analogous to the “sticky ends” created by some restriction enzymes. This
annealing of the right and left ends of lambda to each other explains the occasional to
common appearance of a 27,491 bp band and the partial loss of the 4361 bp band seen in
HindIII digests of lambda (look at the ideal gel and you will see it)! The 23,130 bp left
terminal fragment and the 4361 bp right terminal fragment anneal to each other to form
the 27,491 bp fragment. The loss of the 23,130 fragment is not as obvious since the
27,491 bp and 23,130 bp are very difficult to separate from each other unless the correct
percentage gel is run very slowly and for a long time.