BIO198L Gene Biotechnology Laboratory

1st Quarter SY 2015-2016

Restriction Mapping
Lomugdang, Fiord Jogardy B.1

1Student (s), Subject/Section, School of Chemical Engineering, Chemistry and Biotechnology, Mapua Institute of Technology

ABSTRACT

Restriction enzyme mapping is a powerful tool for the analysis of DNA. This technique relies on restriction endonucleases, hundreds of
which are now available, each one recognizing and reproducibly cleaving a specific base pair (bp) sequence in double-stranded DNA thus
generating fragments of varying sizes. Comparative analysis of fragments generated by cleavage with two different restriction
endonucleases enables the molecular biologist to determine the relative location of particular recognition sequences in the DNA molecule.
The experiments objectives is to use restriction mapping to determine where restriction enzymes cut a piece of DNA and generate a
restriction map of the cDNA isolated.

Keywords: Restriction mapping, cDNA, base pair

INTRODUCTION

A restriction map is a map of known restriction sites within a I. Select the DNA to be cut with restriction enzymes.
sequence of DNA. Restriction mapping requires the use of
restriction enzymes. In molecular biology, restriction maps are 1. Click the arrow on the tube rack to get the DNA selection
used as a reference to engineer plasmids or other relatively short menu.
pieces of DNA, and sometimes for longer genomic DNA. There 2. Select a DNA by highlighting a menu option and
are other ways of mapping features on DNA for longer length DNA releasing the mouse.
molecules, such as mapping by transduction (Bitner, Kuempel 3. When you roll over the DNA tube its contents will be
1981). listed. Each DNA tube contains 1microgram of the
selected DNA at a concentration of 1
One approach in constructing a restriction map of a DNA molecule microgram/microliter as well as 2 microliters of 10x buffer
is to sequence the whole molecule and to run the sequence and 15 microliters of distilled water.
through a computer program that will find the recognition sites that 4. To get another tube, click the arrow again and the menu
are present for every restriction enzyme known. Before will reappear. You can select as many as 10 DNA tubes.
sequencing was automated, it would have been prohibitively 5. To determine the restriction map of a cDNA you will want
expensive to sequence an entire DNA strand. Even today to compare its restriction fragments with those from the
sequencing is overkill for many applications. To find the relative pUC19 plasmid vector alone. For each restriction
positions of restriction sites on a plasmid a technique involving enzyme you choose to use, you should select pUC19
single and double restriction digests is used. Based on the sizes plasmid for one of your DNA tubes.
of the resultant DNA fragments the positions of the sites can be 6. If you make a mistake, drag the tube to the trash can
inferred. Restriction mapping is very useful technique when used underneath the bench.
for determining the orientation of an insert in a cloning vector, by
mapping the position of an off-centre restriction site in the insert
(Dale, Von Schantz, 2003).

II. Add restriction enzymes to the DNA.

1. To select restriction enzymes, click the arrow on the

MATERIALS AND METHODS enzyme cooler. When you make your selection from the

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1st Quarter SY 2015-2016
menu, a tube will appear in the cooler, containing the
selected enzyme.
2. Use the pipettor to transfer restriction enzymes to the
DNA tube.
3. Adjust the volume on the pipettor for the correct amount
of restriction enzyme. Two microliters is an appropriate
amount for a 20 microliter reaction.
4. You can add more than one restriction enzyme to each
tube. The final contents of each tube will be automatically
recorded in your notebook.
III. Incubate the restriction digest
1. After adding the restriction enzymes, drag the DNA tubes
one by one to the heat block.
2. When all tubes are in the heat block, turn it on by clicking
the ON/OFF switch. Set the temperature for 37 degrees
Celsius.
3. Set the timer. Most restriction enzymes will completely
digest DNA within 1 hour. If less time is allowed for the
digestion, then you will get a partial digestion.
4. While the reaction digest is incubating, click here to
access an animation that will show you what's going on
in the tubes.
IV. Add running dye to the digested DNA.
1. When the timer signals the end of the incubation, press
the NEXT button to move down the bench to where the
next rack is located.
2. Drag the tubes, one by one, to the empty rack.
3. To prepare your digested DNA for running on a gel you
must add running dye. The running dye or blue dye is
in the small rack.
4. Use the pipettor to add blue dye to each of your digested
DNAs. For a 20 microliter DNA reaction, add 2 microliters
of blue dye.
V. Load the agarose gel
Experiment 01 Date: August 03 2015
1. You will now use agarose gel electrophoresis to separate
the DNA fragments. Click here to view a movie on how to
prepare an agarose gel.
2. Use the pipettor to transfer 10 microliters of marker DNA
found in the small rack to the first gel well on the left.
When the pipettor tip is over the well, release its contents
by releasing the mouse button.
3. Use the pipettor to transfer 10 microliters of the digested
DNA (to which you have added blue dye) to a different
well of the gel. The contents of each well are revealed
when the pipettor rolls over them and they will be
automatically entered into your notebook.
VI. Run the gel.
1. When all of the wells are loaded, click the NEXT button.
2. Drag the cover onto the gel box. It is correctly positioned
when you hear it click into place.
3. Adjust the voltage by clicking on the voltage/amperage
indicator. 100 volts is an appropriate voltage for this gel.
4. Turn on the power supply by clicking the ON/OFF switch.
A new window will open to give you a good view of the
gel inside the gel box.
5. After a few moments, an ultraviolet light will illuminate the
gel in which Ethidium bromide binds to the DNA
fragments and glows orange.
6. The voltage can be adjusted while the gel is running.
7. You can stop and resume the electrophoresis at any
time. The goal is to get maximum separation of the
fragments without letting any of them run off the bottom
of the gel. Fragments that do run off the end of the gel
are lost.
8. Click on the camera icon to take a picture of the gel. The
picture will be entered intoyour notebook. You can take
up to three pictures of each gel.
9. If you would like to digest another set of DNAs with the
same or other enzymes, close the window to return to the
bench.
10. This may be a good time to save your work by clicking
the SAVE button in the toolbar.
VII. Determine the size of the restriction fragments
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1. To get an estimate of fragment size, compare the position tubes were added with restriction enzyme. The enzyme used was
on the gel of a DNA fragment with the position of the gel BamHI.
marker DNA bands. Those that appear at about the same
place have about the same size. The DNA tubes with restriction enzyme was incubated by placing
2. To get a more accurate size of a restriction fragment, use it on a heat block. This will allow restriction enzyme to completely
digest DNA. The digested DNA was added with a running blue
the ruler to measure the distance from the starting well to
dye in preparation for agarose gel electrophoresis.
each of the gel marker fragments and plot this
information on a piece of graph paper or in a graphing An agarose gel electrophoresis was performed in order to
program. separate the DNA fragments from the DNA tubes. Marker DNA
3. Make the Y axis the logarithm of fragment length and the was loaded to determine the sizes of the fragment from the DNA
X axis the distance migrated. Draw a straight line through tubes. The DNA from the tubes was then loaded to the wells. Th e
the plotted points. gel was run. Finally, the size of the restriction fragments was made
4. Next, use the ruler to determine the distance each and restriction map was made.
fragment from your digested DNA has migrated in the
gel. Electrophoresis Data
5. Plot each of these distances on the graph you made of DNA marker human beta- pUC19 plasmid
globulin cDNA in vector/ BamHI
the gel marker DNA size versus distance and determine
pUC19/BamHI
the approximate size of the fragment and make a table of
distance Size distance Size(bp) distance Size(bp)
the sizes of the fragments generated by each enzyme. (bp)
1.2 23130 3.9 2884 4 2673
VIII. Make a restriction map 2.3 9416 7 266
2.9 6557
1. For each restriction enzyme that you used, compare the 3.4 4361
number of fragments obtained for the cDNA (inserted into 4.2 2322
pUC19) with the number of fragments obtained for 4.3 2027
pUC19 alone. 6 564
2. If the number of fragments is the same it means that this 8 125
enzyme does not cut within the cDNA. If the number of
fragments is greater for the cDNA than for pUC19, it It is easy to determine the unknown size of the restriction
means there is at least one restriction site for that enzyme fragments given the known fragments sizes of the DNA marker.
This will be used to make a restriction map. To get the accurate
within the cDNA.
measurement of the fragment, linear regression was used. Using
3. With the data you generated in this module make a the data of the DNA marker, we get an intercept of 4.76, slope of
restriction map, using the examples as a guide. You will -0.33, and line of best fit of y = -0.33x + 4.76. This equation was
probably have to do additional experiments to make a used to get the accurate sizes of the DNA fragment. This will be
complete map. used to make the restriction map.
4. To determine the sequence of this cDNA using the
Sequencing module, locate the restriction sites that will The sample cDNA (human beta-globulin cDNA in pUC19) was cut
let you subclone fragments of appropriate sizes. For with BamHI and produced two fragments, 2884bp and 266bp. It
manual sequencing you will need fragments that are no tells the combined length of cDNA and pUC9 is 2884 + 266 = 3150
longer than 500 bp. bp. Because the length of the pUC9 is about 2700 bp, it can be
estimated the size of cDNA 3150-2700 = 450bp. Therefore, the
RESULTS & DISCUSSION size of the BamHI site is 266bp and the shorter fragment is the

In the experimentation, the DNA used were human beta-globulin

cDNA in pUC19 and beta-globulin cDNA in pUC19. These DNA

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remaining 184bp of the cDNA still attached to the 2700bp of the
pUC19.
Figure 1. Result from Gel electrophoresis
Figure 2. Restriction Map
Experiment 01 Date: August 03 2015
CONCLUSION
Restriction mapping is used to determine where restriction
enzymes cut a piece of DNA. It can generate a restriction map of
the cDNA you isolated in the cDNA Cloning module or of a PCR
fragment you amplified in the PCR module. It can use the
restriction map to decide which fragments to subclone for
sequencing or for insertion into a protein expression vector.
REFERENCES
Moffatt, B (2006). Course Notes Biology 208.Waterloo, University
of Waterloo.
Dale, J, & von Schantz, M (2003). From Genes to
Genomes.West Sussex: John Wiley & Sons Ltd.
Bitner, R, Kuempel, P (1981). P1 Transduction Mapping of the
trg Locus in rac+ and rac.
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