DNA Restriction Analysis

Ella Beidler
Honors Biology
Period 3
May 24, 2016

Introduction
Restriction enzymes are enzymes that cut DNA molecules at particular places. They play
a big part in recombinant DNA technology. They occur when the enzyme scans a DNA
molecule, looking for a particular sequence, usually four to six nucleotides. Once it finds this
recognition sequence, it stops and cuts the strand (askabiologist.asa.edu). The particular
sequences are called recognition sites, where the enzyme wraps around the DNA and causes a
break in both strands of the DNA molecule. The ability to cut DNA has made it useful for
scientists to continue research and participate in new experiments in gene technology.
Gene technology is the subject of genetic understanding, transferring, modifying, and
testing. Using parts of DNA, scientists are able to conduct research labs of inserting or replacing
DNA into organisms. For instance, scientists have been researching a cure for cystic fibrosis in
humans by adding the certain normal gene to replace the defective one found in most candidates
that hopefully eliminate a cells chance of inheriting the disease. Another example of gene
technology would be the restriction fragment length polymorphism method, also known as
RFLP. RFLP is a widely used test in criminal and paternity cases to compare DNA samples. It
gives scientists the ability to measure repetitions of individual DNA strands which ultimately
proves if a suspect is guilty or if people are genetically related to one another.
In this lab, gene technology was used to analyze DNA using Gel Electrophoresis. This
test is used to separate molecules for the analysis of the molecular components of living
organisms (qwhatis.com). It works by setting a neutrally charged gel into a holding tank. The
holding tank contains edges of the molecule samples and is filled with buffer solution. An
electric current is added that pushes the negatively charged DNA molecules away from the
negative end of the tank to the positive side. A different variation of Gel Electrophoresis is RFLP

Electrophoresis. It is a test to that analyses, a difference in homologous DNA sequences that

can be detected by the presence of fragments of different lengths after digestion of the DNA
samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is
specific to a single clone/restriction enzyme combination (ncbi.nlm.nih.gov). The RFLP
fragments separate along with other molecules in the gel to create a certain pattern based on the
individual DNA. Scientists are able to collect the data and compare samples to match a certain
strand of DNA in many paternity and criminal cases.
The purpose of this lab was to practice with restriction enzymes and Gel Electrophoresis.
The goal of this was to see if different restriction enzymes cut DNA into different sized
fragments or the same fragments. After the data was collected, students were to create a
logarithmic graph with the known data to figure out the other lengths of the DNA fragments that
were created by different reaction enzymes cutting them. The hypothesis of the is experiment
was that the four test tubes of Lambda DNA with the three restriction enzymes BamHi, EcoRI,
and HindIII, and one with no enzyme, will end up with different sized DNA fragments because
the restriction enzymes will cut the DNA in different places. The control group of this
experiment was the tube with no enzyme. The three enzymes were the changed variables while
the length of their fragments were the dependent variables. The fragments of the three enzymes
and the no enzyme were all analyzed and compared.
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

Procedure A
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 the 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
4L
4L
4L
4L

Buffer
5L
5L
5L
5L

BamHI
1L
-------

EcoRI
--1L
-----

HindIII
----1L
---

H2O
------1L

3. Pool and mix reagents by tapping the tube bottom on lab bench, or with a short pulse in
a microcentrifuge.
4. Incubate all reaction tubes for a minimum of 20 minutes at 37 .

Procedure B

1. Add 1L loading dye to each reaction tube. Mix dye with digested DNA by tapping tube
on lab bench, or with a pulse in microcentrifuge.
2. Use micropipette to load contents of each reaction tube into a separate well in the teacher
generated gel, aligned as illustrated in Ideal Restriction Digest of Lambda DNA. Use a
fresh tip for each reaction tube.
a. Steady pipette over well using two hands.
b. Be careful to expel any air in micropipette 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 pipette 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 pipette through bottom of
gel.
Procedure C
1. 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.
2. Turn power supply on and set voltage as directed by instructor. Shortly after current is
applied, loading dye can be seen moving through get toward positive pole of
electrophoresis apparatus.
3. 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 a rate equivalent to approximately
2000 base pairs.
4. Allow the DNA to electrophorese until the bromophenol blue band nears the end of the
gel. The Instructor may monitor the progress of electrophoresis in the students absence;
in that case, omit steps 5 and 6.

5. Turn off power supply, disconnect leads from the inputs, and remove top of
electrophoresis chamber.
6. Carefully remove casting tray and slide gel into staining tray labeled with group name.
Take gel to instructor for staining.
Results

Here is a picture of the ideal gel measurements of the three enzymes tested and the no enzyme
after gel electrophoresis.

Distance Travelled by Fragment Size Cut with HindIII

1.6
1.4
1.2

f(x) = - 0.01x + 1.9

Fragment Size (log kbp)

0.8
0.6
0.4
0.2
0
30

40

50

60

70

80

90

100 110 120 130

Distance traveled by Fragment (mm)

This graph shows the traveled distances of the fragments of HindIII. The equation is the line of
best fit and was used to find the distances of EcoRI and BamHI.

Enzyme
HindIII

Distance
Calculated bp
Actual bp
42mm
-----27, 491
46.5 mm
-----23, 130
60.5mm
-----9,416
70 mm
-----6,557
83.9 mm
-----4,361
115 mm
-----2,322
123 mm
-----2,027
EcoRI
43mm
20,412
24,756
48mm
17,434
21,226
68mm
9,276
7,421
76mm
7,207
5,643
81mm
6, 156
4,878
95mm
3,958
3,530
BamHI
52mm
15,361
16, 841
56mm
13,545
12,275
68mm
9,276
7,233
71mm
8,439
6,527
76mm
7,207
5,505
This graph shows the distance of the fragments of the three enzymes measured on the photo. It
also shows the final calculated base pairs of the enzymes the students tested as well as the actual
base pairs of the fragments which was given to students by the teacher.

Discussion
Based on the data collected from the experiment, the hypothesis was proven correct. Each
enzyme had different lengths compared to each other which proves that restriction enzymes do
cut DNA at different locations. The reason that some fragments traveled farther in the gel is
because smaller fragments are more likely to fit through the gel than larger ones, giving it more
distance. The gel used in the experiment did not show the lengths of the fragments tested. This
error is believed to be caused by a mistake during the staining process or the UV light that was
used to analyze the results. Since the final results were not evident, students based their

measurements off of the ideal gel image. Measuring the results from a photo brought more
chances for mismeasurements. Students could have measured at the wrong edge or could have
messed up the measurements by rounding up their final answers to translate the sizes to base
pairs.
Works Cited
https://www.askabiologist.asu.edu/restriction-enzymes
http://www.qwhatis.com/what-is-gel-electrophoresis/
http://www.ncbi.nlm.nih.gov/probe/docs/techrflp/
DNA Restriction Analysis Lab Manual