Download DNA -- Teacher Preparation Notes

DNA -- Teacher Preparation Notes
by kllni!'cr Duherty nil,,) D,. Ingrid W~ldru<l. D~parlmcn\ of Blolugy. Uni,..,r,;t)' uf Pcll11.'iylvania, 2007'
As b<lckground for this activity, students should know thm DNA is the genetic material and DNA
is contained in chromosomes inside the nucleus inside a cell. We recommend that this activity
be followed by our activity, "From Gene 10 Protein -- Transcription and Transl'lIion", which
continues discussion of the importance of the base-pairing rule and explains how the sequence of
nuclcolidcs in a gene dctcnnincs characteristics such as sickle cell anemia.
Equipment and Supplies:
Sports Drink like Gatorade (10 mL per srudcnt)
Liquid dish soap (0.25 mL per student)
Meal Tenderizer (a pinch per s\lldent)
70-95% isopropyl or ethyl alcohol (4 mL per student)
String for necklacc (2,5 n pcr student)
3 oz, Dixic C~IPS (1 pcr student)
Tub of ice, freezer, or refrigerator (I)
Tllb for dirty test tubes (1)
Ble<lch (1%1 bleach solution to sterilize test tubes)
Small test tubes (tubes need to hold a minimum of IS mL) (1 per sludent)
Test tube raek ([ per gl"oup)
0.5- 1.5 I11L lliptop microcentrifuge tubes (l per studem)
Transfer pipcttes (I per group)
Gloves (1 per student)
Items aho"'e not found locally can be purchased from Carolina Biolo~jcol among other
places:
73-0006
12 )( 75 mill Test Tubes
524.40 Pack 01'72
19-9684
1.5·mL Mieroeentrifuge Tubes
543.65 Pack or 1,000
21-5240
73-6984
O.5-mL Mierocenlrifuge Tubes
Pipcltcs, Graduatcd, Small
571.00 Pack or 1,000
54.35 Pack of! 00
Before Class:
I. Cut string imo 2.5 n scgmcnts.
2. Set up a bucket ofiee to keep alcohol cold or put alcohol in the freezer/refrigerator umil
needed.
3. Pour a smull cup sports drink for each slUdent.
During class:
1. Distribute cups of sports drink to each studcnt. It is important for each student to swish lhe
drink in his or her mouth for al least a minute in order to obtain enough cheek cells.
2. Distribute a tcst tube rack willI one tcst lubc per student to eueh group. Distribute one glove
to each studenL. Pass around the soap and meat tenderizer. Altematively, you cun have a
station somewhere in thc cl<lssroom where the students can access the soap etc.
3. Ancr at least 10 minutes (when the students have completed the "DNA structure" portion of
thc workshcet), pass <lround the cold alcohol <lnd pipettes.
4. After 10 minutes (when the students have completed thc "DNA replica\ion" portion of thc
worksheet), distribute one microcentrifuge tube and piece of string to cat:h studcnt.
I
These leal'her preparation nOles and the relaled student handoUL are available at
h!1p:f/~l'r<:nd in .brYIl m:lwr. ~d (l1~c;
cd (11"':1 ld ron/.
In tp: Ilwww. ~ k\-.\l.: ielll.:e .(<lln'p Jasli (-I abo ralory-via15-P-7878. hITIll
5. Assist students' transfer of their DNA to their microcentriCuge whes using the pipettcs. It
hclps to twirl the DNA <lrolll1d the end of the pipette to get a large wad together before
sucking thc DNA into the pipette. Warn the students to be gentle \vhilc pipetting so they do
nOl dUllloge the fragilc strands of DNA. lnexpcrieneed pipelLers have a tendency to blow air
into (he liquid and slick up and expel the DNA several times in the test tube before
IrIll1sferring il to thc microccntrifugc tube; this tends to break the DNA strands.
6. Put on a pair of glovcs <.Ind collect the test tube racks Cram the students. Pour lest tube
contcnts out down thc sink, rinsc the testtube1, and place them in a tub of I % bleach solution
for 10 minutes 10 sterilize them for the next class. (You lllay \vant to assign this job to a
studenl).
7. Remove test lUbes from ble<.lch water and in\'en them in [he racks to dry for the next class.
8. RelUm the alcohol to the icc bucket or freezer/refrigerator.
Supplemenl:J1 Acti"'ity
You mllY also wOlllto cxtrnct DNA from other organisms, such as peas or strawberries. to show
your students th<.ll DNA from different organisms·looks the same. This is also helpful to provide
DNA to Ill<.lke a nccklacc for a student whosc own DNA extraction did not work (e.g. due to
insufftcient cheek cell collection). For instnlctions to extract DNA from other sources scc "How
to Extract DNA from Any Living Thing" at the Genetic Science Learning Center
http://l eorn. genel] cS.lItah .cd u!lln i1ShlCI ivilll.:s!c'l: tract ion!
AdditionallnforlllaLioll
The proteascs in thc meat tenderizer not only digest hislOnes (the DNA wrapping proteins), but
also break down rhc cnzymcs which digest DNA.
Cold alcohol hclps to precipitate the DNA moleeules by reducing the temperature and
dehydrating the salty soapy solution of DNA immediately under the alcohol layer. The high salt
concentration, from the sports drink and meat tenderizer, is also imponanl since DNA molecules
are negatively charged and the salt neutralizes [he repulsion among the negatively charged
strands of DNA and allows thc DNA to clump logether.
Genetic Sciencc Learning Center has a Frequenlly Asked Questions page including answers (0
questions about Trouble-shooting, Understanding the Science behind the Protocol, Comparing
the ON A Exlracted from Di fferenl Cell Types, and Real-life Applications of the Science of DNA
Extrnction. http://learn.genclics.lIlah.edu/units!acti vi ties!eXtraclion/fag.c fm
For more delailed infonnation about DNA extraelion and helpful figures for explaining how
detergents break down lipids see hllp://learn.genetics.utah.edu!units!activities!extraction! and
h((p:!!www.biorad.colll (seareh "genes in a boule;').
DNA Teaching Points
DNA is a nucleic acid made of two strands ofnueleotides wound together in a spiral called a
double helix.
Each nucleotide is composcd ofa five carbon sugar molecule known as deoxyribose, a
phosphate group, and one of four different nitrogenous bases: adeninc, thymine, guanine, and
cytosine.
.
Nuclcotides in the two strands follow the base-pairing rule: A with T and G with C.
During DNA replication DNA polymerase produccs two identical DNA double helixes.
DNA polymerase forms a new DNA strand, using the old DNA strand as a templatc, by
adding complcllll.:lltary base pair Ilucleolides to a growing DNA strand.
The DNA ofdiflerent org,lllisms differs only in thc sequence of nucleolides.
•
DNA
B"sed on find adapted from the GenBlic Science Learning Cenler's
"How to Extract DNA Irom Any Living Thing" (http://learngenelics,utah.edulunitslactiviliesfexlraction/)
and BioRad's "Genes in a bollle" (hltplfWoNw.bio-rad.comltmc upload/lileralurel5413314110034B,pdf).
by Jennifer Doherty and Dr. Ingrid Waldren, Department of Biology. University of Pennsylvania. © 2007'
Furtfler edited for the CaMSP POSE Project
Today you will extract DNA from some of your cells and learn more about DNA.
Why is DNA so important in biology? What is the function of DNA?
Where is DNA found in our bodies?
Draw a simple diagram of a cell, showing the cell membrane and the DNA in chromosomes
surrounded by a nuclear membrane.
Extracting DNA from Your Cells
Cells from the lining of your mouth come loose easilY, so you will be able to collect cells containing
your DNA by swishing a liquid around in your mouth.
The cells from the lining of your mouth also come off whenever you chew food. How do you think
your body replaces the cells that come off the lining of your mouth when you eat?
To extract DNA from your cells, you will need to separate the DNA from the other types of biological
molecules in your cells. What are the other main types of large biological molecules in cells?
-
You will be using the same basic steps that biologists use when they extract DNA (e.g. to clone DNA
or to make a DNA fingerprint). You will follow these 3 easy steps to extract the DNA:
Detergent
eNzymes (meal tenderizer)
Alcohol
Getting Your Sample of Cells
Obtain a cup with sports drink. You will need to get thousands of your cheek cells in the sports drink
in order to extract enough DNA to see. Therefore you should swish the sports drink around in your
mouth vigorously for at least one minute. Then spit the drink back into the cup. You do not want to
use a lot of sports drink because you do not want a lot of fluid to dilute your cheek cells.
Step 1: Detergent
Add a small amount of detergent to a test tube (a drop is plenty). Now carefully put the drink
containing your cheek cells into the test tube with detergent until the tube is half full. Use a plastic
pipette to move the drink/cheek cell mixture
Why am f adding detergent?
To get the DNA out of your cheek cells you need to break open both the cell membranes and the
nuclear membranes. Cell membranes and nuclear membranes consist primarily of lipids. Detergents
break up lipids (Fats and Oils). This is why you use detergents to remove fats (which are lipids) from
dirty dishes. Shampoo also has detergents to help remove excess oil from your hair. Adding the
detergent to you cheek cell solution will break open the cell membranes and nuclear membranes and
release your DNA into the solution. The detergent we use is called Sodium Lauryl Sulfate and can be
found in many brands of shampoo. Scientists use the same chemical to help dissolve lipids and
breakup (or denature) proteins.
Step 2: Enzymes
Add a pinch of enzyme (meat tenderizer) to your test tube. With your gloved thumb (or palm)
covering the top of the test tube; gently invert the tube five times to mix. Let the mixture sit for at least
10 minutes. While you are waiting, you will learn about the structure of DNA. Remove your glove
and throw it in the garbage.
Why am I adding enzymes?
The nucleus of each of your cells contains multiple long strands of DNA with all the instructions to
make your entire body. If you stretched out the DNA found in one of your cells, il would be 2-3
meters long. To fit all of this DNA inside a tiny cell nucleus, the DNA is wrapped tightly around
proteins. The enzyme in meat tenderizer is a protease, which is an enzyme that cuts proteins into
small pieces. As this enzyme cuts up the proteins, the DNA will unwind and separate from the
proteins.
The protease in meat tenderizer actually comes from either Papaya or Pineapple (it all depends on
the brand), but animals also make proteases. Where in your body do you think you make protein­
cutting enzymes?
DNA Structure
As you can see in the figure below, DNA consists of two strands of nucleotides wound together in a
spiral called a double helix. Each nucleotide contains a phosphate and a sugar molecule called a
deoxyribose (which explains why the complete name for DNA is deoxyribonucleic acid). Each
nucleotide also has one of four different nitrogenous bases: adenine (A), thymine (T), guanine (G),
and cytosine (e).
"­
c::::J .......0. w
c::::J
Cyl--.o ICJ
L
c:::J
r......- iT!
~
(iooNono
Ie)
(Adapted from Figure 9.4 in Biology by Johnson and Raven)
The drawings below show a very small section of the DNA double helix from three very different
organisms: a plant, a mammal, and a bacterium. Each strand of DNA shown contains five
nucleotides, each with a:
S = five carbon sugar molecule called deoxyribose
P = phosphate group
A = adenine, C = cytosine, G = guanine, or T = thy'!)ine, the DNA nucleotide bases
'>-EA:r(~T}-<
,
p
A
T
AT'
p
~>-jJTD)~AQ--< ,
, >-j[!A'l(JTQ--<,
Plant
Mammal
Bacterium
(From BioRad's 'Forensic DNA fingerprinting kir h\lpllwww.bio-rad.comJffilc_uploadILiler<lwre/1252514006096G,pdf)
You can see that the phosphate from one nucleotide is bonded to the sugar in the next nucleotide to
form the backbone of each strand in the DNA molecule. The bases of the nucleotides in each strand
of DNA extend toward each other in the center of the DNA double helix molecule. A crucial aspect of
DNA structure is the base-pairing rule: A in one strand always pairs with T in the other strand, and G
in one strand always pairs with C in the other strand. You will see later that this base-pairing is
crucial for the cell to make new copies of each DNA molecule in preparation for cell division.
1. Compare the sugar-phosphate arrangement in the backbone of the DNA from the plant, the
mammal and the bacterium. Are there any differences?
2. Which bases are present in the DNA of the plant? The mammal? The bacterium?
Are the same bases present in all three cases?
Are the bases in the same order?
3. Describe the pattem of base pair matching for the two strands in the plant's DNA. In other words,
which types of bases are paired together? Does the DNA from the mammal follow the same
base-pairing rule as the DNA from the plant? Is base-pairing the same or different in the DNA of
the bacterium?
Which characteristics are similar in the DNA of plants, mammals and bacteria? What is the only
characteristic that differs between these segments of DNA from a plant, a mammal and a bacterium?
These observations illustrate the similarity of the basic structure of DNA in all living organisms. The
genetic differences between plants, mammals and bacteria are due to differences in the sequence of
bases in their DNA.
Step 3: Alcohol (after the 10 minute wait)
Using a pipette, slowly add cold rubbing alcohol into the test tube; let the alcohol run down the side of
the test tube so it forms a layer on top of the soapy liquid. Add alcohol until you have about 2 em of
alcohol in the tube. Alcohol is less dense than water, so it floats on top. 00 not mix or bump the
test tube for 10 minutes. DNA molecules will clump together where the soapy water below meets
the cold alcohol above, and you will be able to see these clumps of DNA as white strands.
Why am I adding alcohol? The cold alcohol reduces the solubility of DNA. When cold alcohol is
poured on top of the solution, the DNA precipitates out into the alcohol layer, while the lipids and
proteins stay in the solution.
After about 10 minutes you should be able to see white strands (or clumps) of your own DNA. A
toothpick or a stir stick can be gently used to pull more DNA out of the bottom detergent layer into the
alcohol layer.
Question: Why does DNA or genetic material make living things so different from non-living things?