Restriction Analysis of DNA

Lawren Szilagy
Honors Biology
Period 5

Introduction
Restriction enzymes are enzymes used to cut DNA at specific nucleotide sequences into
smaller fragments. Different restriction enzymes recognize and cut different DNA sequences.
Restriction enzymes come into contact with a DNA sequence with a recognition site and wrap
around the DNA, causing the DNA to break into fragments. Scientists and researchers use
restriction enzymes to execute almost any process that involves manipulating, analyzing, and
creating new combinations of DNA sequences. Among the many new combinations are DNA
cloning, hereditary disease diagnosis, paternity testing, forensics, genomics (e.g., the human
genome project), epigenetics, genetically modified organisms, and biotechnology (nature.com).
Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms
may be differentiated by analysis of patterns derived from cleavage of their DNA. If two
organisms differ in the distance between sites of cleavage of a particular restriction
endonuclease, the length of the fragments produced will differ when the DNA is digested with a
restriction enzyme (kau.edu.sa). RFLP is used in paternity and criminal cases to determine the
source of a DNA sample, as well as in determining the disease status of an individual and
measuring recombination rates, which can lead to a genetic map. In criminal and paternity cases,
scientists use variable regions of DNA to generate a DNA profile of an individual using blood
samples or samples from crime scene evidence. They then find markers in the DNA sample by
designing small pieces of DNA probes that seek out and bind to a complementary DNA sequence
in the sample. A series of probes bound to the DNA sample creates a distinctive pattern for an
individual, that of which scientists compare to determine whether a suspects sample matches
the evidence sample.

Gel electrophoresis is a technique used to separate charged molecules according to size.

Gel electrophoresis separates macromolecules such as DNA, RNA, and proteins according to
molecular size so that researchers can further analyze them. After a gel is prepared, DNA
samples are loaded onto it and an electric current is applied. The negatively charged DNA is
attracted to the positive electrode. As the pieces of DNA move through the gel, they feel
resistance, resulting in larger pieces to move at a slower rate than smaller fragments, allowing for
the separation of different sized DNA fragments.
The purpose of the lab is to observe if different restriction enzymes cut DNA into
different sized fragments or the same sized fragments. The students also want to gain experience
with restriction enzymes and gel electrophoresis, as well as with creating a logarithmic graph
using known data to figure out other lengths of DNA fragments cut by different restriction
enzymes. The independent variables in this experiment are the DNA samples with the restriction
enzymes, and the dependent variable in this experiment is the distance the DNA cut by different
restriction enzymes travels. The control group in this experiment is the DNA with no restriction
enzyme added to it. It is hypothesized that if restriction enzymes are applied to DNA samples,
the enzymes will cut the DNA fragments into different lengths.

Materials

Agarose Gel
TBE Buffer solution
Lambda DNA
Restriction Enzymes (EcoRI, BamHI, HindIII)
Micropipettes
Micropipette tips
Hot plate
Eppendorf reaction tubes
50 mL beakers
1000 mL flask
Electrophoresis chamber
Graduated cylinder
Microcentrifuge
Vortex
Ethidium bromide stain
Loading dye
Gloves
Goggles
Staining trays
Ultraviolet light source
Sharpie

Procedure
1. Label four 1.5-mL tubes, in which you will perform restriction reactions: B for BamHI, E for
EcoRI, H for HindIII, and for no enzyme.
2. Use table below as a checklist while adding reagents to each reaction. Read down each
column, adding the same reagent to all appropriate tubes; use a fresh tip for each reagent. All
groups share the same BamHI, EcoRI, HindIII enzymes at a central station.
Tube
B
E
H
__

DNA
4 L
4 L
4 L
4 L

Buffer
5 L
5 L
5 L
5 L

BamHI
1 L
__
__
__

EcoRI
__
1 L
__
__

HindIII
__
__
1 L
__

H20
__
__
__
1 L

3. Pool and mix reagents with a short pulse in a microcentrifuge.

4. Incubate all reaction tubes for a minimum of 20 minutes at 37C.
5. Add 1 L loading dye to each reaction tube. Mix dye with digested DNA with a pulse in
microcentrifuge.
6. Using teacher generated gels, use micropipet to load contents of each reaction tube into a
separate well in gel, aligned as illustrated in Ideal Restriction Digest of lambda DNA. Use a
fresh tip for each reaction tube.
a. Steady pipet over well using two hands.
b. Be careful to expel any air in micropipet tip end
before loading gel. (If air bubble forms cap over well, DNA/loading dye will flow into
buffer around edges of well.)
c. Dip pipet tip through surface of buffer, position it over the well, and slowly expel the
mixture. Sucrose in the loading dye weighs down the sample, causing it to sink to the
bottom of the well. Be careful not to punch tip of pipet through bottom of gel.
7. Close top of electrophoresis chamber and connect electrical leads to an approved power
supply, anode to anode (red-red) and cathode to cathode (black-black). Make sure both
electrodes are connected to same channel of power supply.
8. Turn power supply on and set voltage as directed by instructor. Shortly after current is applied,
loading dye can be seen moving through gel toward positive pole of electrophoresis apparatus.
9. The loading dye will eventually resolve into two bands of color. The faster-moving, purplish
band is the dye bromophenol blue; the slower-moving, aqua band is xylene cyanol. Bromophenol

blue migrates through gel at same rate as a DNA fragment approximately 300 base pairs long.
Xylene cyanol migrates at a rate equivalent to approximately 2000 base pairs.
10. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the gel.
11. Turn off power supply, disconnect leads from the inputs, and remove top of electrophoresis
chamber.
12. Carefully remove casting tray and slide gel into staining tray labeled with group name. Take
gel to instructor for staining.

Results

The picture above is an ideal gel showing DNA cut by three different restriction enzymes.

Distance Travelled by Fragments Cut with HindIII

1.6
1.4
1.2

f(x) = - 0.01x + 1.89

1
Fragment Size (log kpb)

0.8
0.6
0.4
0.2
0
30 40 50 60 70 80 90 100 110 120 130
Distance Travelled by Fragment (mm)

HindIII

EcoRI

BamHI

Dis. (mm)

Act. bp

Dis. (mm)

Cal. bp

Act. bp

Dis.
(mm)

Cal. bp

Act. bp

42

27,491

43

19,413

24,756

46

17,636

16,841

46

23,130

46

17,636

21,226

52

14,555

12,275

57

9,416

63

10,235

7,421

63

10,235

7,233

66

6,557

70

8,181

5,804

67

9,005

6,527

81

4,361

77

6,539

4,878

72

7,674

5,505

112

2,322

91

621

3,530

120
2,027
The graph above depicts the distance travelled in millimeters by fragments cut with HindIII.

The table above shows the distance travelled in millimeters and the base pair length for each
fragment cut by different restriction enzymes.

Discussion
The hypothesis was correct, as restriction enzymes cut DNA at different locations.
Smaller fragments travel through the gel more quickly and therefore further than larger
fragments, which travel more slowly. This resulted in the DNA being separated by size. The
students gel was not viewable. This could have been a result from an error in the staining
process or an error with the UV light. This resulted in the student using a picture of an ideal gel
to take measurements. There could have been errors in measuring the ideal gel and therefore
errors in calculations.

References
DNA Restriction Analysis Lab Manual
http://biotechlearn.org.nz/themes/dna_lab/restriction_enzymes
http://biotechlearn.org.nz/themes/dna_lab/gel_electrophoresis
http://www.yourgenome.org/facts/what-is-gel-electrophoresis
http://futurescienceleaders.org/researchers2012/2013/03/how-does-gelelectrophoresis-work/
http://www.kau.edu.sa/Files/0002923/Files/18591_Restriction%20Fragment
%20Length%20Polymorphism.pdf
http://www.nature.com/scitable/spotlight/restriction-enzymes-18458113
http://www.forensicprofiles.com/dna.html