Research into the
genetic origins of
obesity
Extract your own DNA
Experiment workshop
PROTOCOL
Introduction
The incidence of obesity is generally increasing
all over the world. Scientists and doctors are
worried about the accompanying threat to
health and are trying to understand why people
are becoming obese in order to be able to design
strategies for treatment and prevention.
In some cases, obesity can lead to type 2 Diabetes Mellitus (DM2). Diabetes is a disease which
causes high levels of glucose in the blood.
People who are overweight have more fat
around the cells and this makes it more difficult
for the body to use insulin properly. Insulin is a
hormone that makes the cells in the liver, the
muscles and the adipose tissue take up glucose
from the blood, causes the glucose to be stored
in the liver and muscles in the form of glycogen
and stops the use of fat as a source of energy.
Key data on obesity and the
overweight:
• Obesity has more than doubled
worldwide since 1980.
• In 2008, 1.5 billion adults aged 20 and
over were overweight.
• 65% of the world’s population lives
in countries where overweight and
obesity are one of the leading causes of
mortality.
• About 43 million children under 5 were
overweight in 2010. (data from the UN,
March 2011)
Family history is an important factor in terms
of being predisposed to obesity and to type 2
diabetes. Having family members who are obese
and who have this type of diabetes considerably
increases the risk of developing the disease.
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Research into the genetic origins of
obesity
and adipose tissue. The rats’ DNA is sequenced
to obtain the linear order of the nucleotides.
In order to study the potential risk associated
with the hereditary factors for type two diabetes
mellitus, scientists are basing their research
on DNA. They are studying DNA samples from
obese individuals and diabetics and are comparing them to samples from normal individuals to
look for differences.
This line of research has already identified a
gene, given the name DOR (Diabetes and
Obesity Regulated), which behaves in a different
way in diabetic and obese rats than in normal
rats (expressed to a lesser degree).
The research is first being carried out on rats.
The DNA is extracted from their muscle cells
Chromatid
In order to sequence these genes, the scientists
first have to extract the DNA.
Cell
Telomere
Centromere
Nucleus
Telomere
Histones
Base pairs
DNA
(double helix)
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What is DNA?
DNA or deoxyribonucleic acid is an organic
molecule responsible for storing the genetic
information which all living organisms, except
for some viruses, require in order to develop and
function.
Where is it found?
It is found in all living organisms. In bacteria,
the DNA is found in the cytoplasm, while in
more complex organisms, like plants, animals
and other multicellular organisms, most of it is
in the nucleus. DNA is compacted into chromosomes which hold the codes for the essential
instructions to allow an organism to develop and
be able to live. These instructions are the genes,
which store the information and are responsible
for transmitting it to descendants.
What’s it made of?
DNA is a molecule composed of repetitions
of three types of molecules: a nitrogen base,
a phosphate group and a pentose. Each unit
formed by these three types of molecules is
called a monomer and, because DNA has lots of
repeated monomers, we can call it a polymer.
Each of these monomers is what we call a nucleotide. The nucleotides contain different bases
(known as A, C, G, T) ordered in sequence to
form a kind of barcode.
Thymine
Adenine
5’-end
3’-end
Phosphate
Deoxyribosephosphate
backbone
Cytosine
3’-end
Guanine
5’-end
Deoxyribose
(pentose)
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Objectives
Have you ever wondered how the police
extract DNA from samples they find at
crime scenes?
How do scientists separate the DNA
from cells and isolate it from among
the lipids, proteins, carbohydrates and
salts?
Our objective is to extract DNA from our own
cells quickly and easily
Method
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Getting the sample
The first step is to
obtain the cells from
which we’ll extract the
DNA. These cells can
be obtained really easily
by scraping the tongue
and the inside walls of
the mouth.
2
other organic molecules in our sample also degrade. One of these is an enzyme called DNase;
its function is to cut up the DNA.
3. Neutralising the charge on the DNA: this
is done using a salt with many different uses:
sodium acetate (NaAc) The Na+ ions bind to the
phosphate groups in the DNA which are strongly
negatively charged.
Releasing the contents of the cells
Right away, we start processing our sample in
the following order:
1. Lysis: this is the process that breaks open
the cell and nuclear
membranes. The membranes are broken open
by detergents and the
DNA is then released
into the solution.
2. Decompacting DNA:
we use Proteniase K,
an enzyme that degrades proteins that
were bound to the DNA
and kept it compact. So
when it acts, the DNA
is de-compacted and
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Precipitating the DNA
Before adding
ethanol
After
The final step is the
precipitation of the DNA,
which makes the invisible visible.
We use ethanol for
this. DNA is soluble in
water, but when we add
ethanol, it separates and
precipitates. After adding the ethanol, we see
Precipitated DNA
white strands starting to appear in suspension.
This is our DNA.
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Necessary equipment
and material
Laboratory instruments and equipment
20 to 200 μl micropipette
1 to 5 ml micropipette
Timer
Disposable materials
Loop for scraping inside the mouth
15 ml Falcon tube
Plastic Pasteur pipettes
Micropipette tips
Tips for larger
micropipette
Permanent marker
Gloves, lab coat and goggles
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Proteinase K
Reagents and solvents
-20ºC
NaAc
Proteinase K solution
Lysis solution made from
salts and detergents
Sodium acetate solution
(NaAc)
Cold ethanol (keep in
freezer at -20ºC)
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Procedure
A
Extracting the epithelial cells
The first step is always to get a good sample. We do this by taking samples from the mouth. There
are lots of dead cells ready to come off there and they contain a lot of DNA.
1
Prepare a Falcon
tube with 1 ml of
lysis solution.
Leave the tube open
so the next steps are
easier.
2
Scrape hard inside
your mouth for 2
minutes, dragging
the loop end across
the insides of your
cheeks and especially
across the top of your
tongue. That way,
you’ll get enough
cells.
Put the scraper into
the Falcon tube with
the lysis solution and
mix it a bit.
1 ml of lysis solution
Take the scraper
out of the tube and
discard it right away
(it’s important not to
put it back in your
mouth).
3
Suck your cheeks to make saliva to
help drag the cells that have come off
the epithelium.
Collect the saliva with a Pasteur
pipette and put the whole contents in
the tube with the first sample.
4
Put the cap tightly on the tube and mix it
by turning it up and down.
The more saliva, the
more DNA we’ll have!
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B
Releasing the contents of the cells
1
2
Add 20 µl of Proteinase K to cut up
the proteins, resting the pipette on the
wall of the tube and letting it flow in.
C
Put the cap tightly on the tube and
mix by turning it up and down. Leave it
to incubate for 10 minutes.
Precipitating the DNA
1
Calculate the approximate amount of volume (V) collected by checking the scale on the tube.
Enter the volume here:
V=____________ml
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2
Add a tenth of the volume (V/10) of NaAc
solution to the sample.
For example, if you have 1 ml of initial
volume, add 100 µl of sodium acetate:
1 ml=1000 µl 1000 µl/10=100 µl that
needs to be added.
Do your calculations here:
Cap the tube tightly and give it a good
shake up and down.
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4
Add three times the initial volume (Vx3)
of cold ethanol.
Turn it up and down very slowly to mix it.
Your DNA will appear as whitish
precipitate.
For example, if the initial volume of the
sample was 1 ml, you need to add 3 ml
of ethanol:
1 ml x 3 = 3 ml ethanol
Do your calculations here:
Precipitated DNA
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Results and Conclusions
1 – At the end of the session, what did you see? What finally caused that result? Why?
2 – What would have happened if you’d done the last precipitation step with cold water
instead of ethanol?
3 – If you hadn’t done the first lysis step, what do you think would have happened?
4 – Why do you think it’s important to add the Proteinase K? Draw a picture of what you think
happened when you added it.
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5. Which of the components neutralised the charges? Why do you think that step is
important?
6. Do you think this protocol would work with a hair? What about vegetable cells?
7. There is a similar protocol for DNA extraction that uses salty water, washing-up liquid and
ethanol. Match these components with the ones we used in this session.
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Appendix 1
Safety precautions
Inform yourself
Find the safety features in the laboratory or the
place fitted out for experiments (extinguishers,
shower or bath, exits, etc.). Read the instructions carefully before doing an experiment. Don’t
forget to read the safety labels on reagents and
apparatus.
Wear suitable clothing
Gloves, lab coat and protective goggles.
General rules
Smoking, eating and drinking are prohibited
in the laboratory or place fitted out for experiments. Work in an ordered, clean, unhurried
fashion. If a product gets spilled, clean it up immediately. Always leave the material clean and
tidy. Never use equipment or apparatus without
knowing exactly how it works. Wash your hands
before leaving the laboratory.
Handling glass
Protect your hands when handling glass materials and be aware of the temperature – you can’t
tell whether it’s hot or cold just by looking at it.
If the glass is cracked, don’t use it.
Chemical products
Never use any bottle of reagent that has no
label or is not properly identified. Do not smell,
inhale, taste or touch the chemical products.
Never pipette with your mouth. Wear gloves and
wash your hands frequently when using toxic
or corrosive products. If they come into contact
with your eyes, wash them out immediately with
lots of water. Do not put reagent containers near
a flame. Do not heat up inflammable liquids.
Carry bottles by holding them by the bottom,
never by the top.
Disposing of waste
Dispose of solid waste and liquid waste that so
requires in duly identified special containers. If
in doubt, ask your teacher. Never put solid waste
down the sink.
Remember
If an accident happens, tell your teacher immediately.
SPECIFIC PRECAUTIONS FOR THIS WORKSHOP
• SDS:
toxic if ingested, inhaled and in contact with the skin.
• Tris-Base:
• HCl:
toxic if ingested, inhaled and in contact with the skin. Irritant.
toxic if ingested, inhaled and in contact with the skin. Irritant and corrosive.
• Sodium acetate:
• Proteinase K:
• Ethanol:
toxic if ingested, inhaled and in contact with the skin.
toxic if ingested, inhaled and in contact with the skin.
inflammable.
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Appendix 2
Procedures for preparing the solutions
Recipe for lysis solution (in view of the toxicity of some of the compounds at high concentrations, we
recommend that this solution be prepared beforehand by the teacher. Wear a mask while preparing
the solution. Once dissolved, a mask is no longer necessary).
For 50 ml:
- NaCl = 0.292 g
- SDS = 5 ml 10% SDS
- Tris HCl = 2.5 ml pH 8
Recipe for NaAc
For 40 ml:
- 9.84 g NaAc. Add HCl until achieving a pH of 5.2
Recipe for Proteinase K
- 100 μg/ml (keep frozen)
Appendix 3
References for buying the reagents and some of the necessary materials
NAME
REFERENCEMANUFACTURER
NaCl
71381
Fluka-Sigma
SDS
71725
Fluka-Sigma
Tris-Base
93350
Fluka-Sigma
HCl
320331
Sigma-Aldrich
NaAc
S2889-250G
Sigma-Aldrich
Proteinase K
P6556-100mg
Sigma
Ethanol
141086.1214
Panreac
Sterile culture loop
Sanilabo
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Keep investigating Xplore Health!
Researchers who have contributed to the contents: Lorena Valverde, Researcher at Universitat de Barcelona
DEVELOPED BY
FUNDED BY
This work is covered by a Creative Commons Attribution-NonCommercialNoDerivs 3.0 Unported licence deed. To view a copy of the licence, visit
http://creativecommons.org/licenses/by-nc-nd/3.0/
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