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DNA Science
A Hands-On
Workshop
Illustration from the booklet, Alzheimer’s Disease: Unraveling the Mystery,
available from the ADEAR Center. Illustrator: Christy Krames.
Spring 2007
Dr. Sarah C.R. Elgin
Dr. Mark Johnston
Dr. Elena Gracheva
Dr. Kelly J. Wright
Supported by a grant from Howard Hughes Medical Institute
to Washington University
Lab manual by Dr. April Bednarski
2
Table of contents:
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Laboratory Schedule
4
Lab safety & Learning to Use Micropipeters
5
Spooling purified DNA
6
Models of DNA Structure
11
PCR and genetic variation in humans. Alu. Part 1.
15
PCR and genetic variation in humans. Alu. Part 2.
23
Sterile techniques and transforming bacteria with DsRed. 25
Learning to clone DNA. Part 1
31
Learning to clone DNA. Part 2.
39
Learning to clone DNA. Part 3.
43
Chymotrypsin enzyme activity assay.
48
Yeast genetics
53
Reebops: Inheritance of traits.
59
Can sunscreen stop mutations?
65
Learning to use bioinformatics tools.
71
Glossary.
95
Website list for Bio 280
101
Genetic glossary
104
3
Laboratory Schedule
Biology 280
Spring 2007
Date
1/19
1/26
2/2
2/9
2/16
2/23
3/2
3/9
3/23
3/30
4/6
4/13
4/20
4/27
Lab
Lab Safety
DNA spooling
DNA models
Alu PCR, part 1.
Transforming bacteria with DsRed, part 1.
Alu PCR, part 2.
Transforming bacteria with DsRed, part 2.
Yeast cloning, part 1.
Yeast cloning, part 2.
Yeast cloning, part 3.
Chymotrypsin activity assay.
Intro to bioinformatics.
Tour of the GSC.
Yeast genetics. Reebops.
Sunscreen, part 1.
Sunscreen, part 2.
Bioinformatics: DNA sequence comparative analysis.
Bioinformatics: protein structure.
Bioinformatics: human disability.
Paper reports.
4
Lab safety & Learning to Use Micropipeters
Pipetmans™∗ are instruments used to accurately transfer small volumes (1µl to
1ml) of solution. Because of their accuracy, ease of use, and convenience in
sterile techniques, they are practically universal lab tools. In this week’s lab, you
will first learn how to properly use these instruments. After you feel comfortable
accurately transferring small volumes onto a filter paper and from tube to tube,
you will learn how to transfer liquids into a gel. These techniques will be used
throughout our course.
Use the picture below and the pipetmen on your desk to identify the
following parts. Label each part.
Barrellower
The working end of the pipetman; a disposable tip is seated on the
end of the barrel before each use.
The part located at the top that is pressed and released to withdraw
and expel liquid.
Tip ejector buttonUsed to remove a tip from the barrel without direct handling of the
disposable tip.
Volume settingDisplays the volume the pipetman is currently set to deliver.
Volume adjustment knobRotated to change the digital volume setting.
Plunger-
“Pipetman” is a brand name. The generic name for this instrument is “micropipet". In a research lab you
are likely to hear these terms interchangeably.
∗
5
At your lab bench you will find three pipetmans; each one is appropriate
for a specific volume range. If you look at the dot on the top of the plunger of
each pipetman, you will see a number that represents the maximum volume, in
microliters (µl), that can be transferred by that pipetman. The minimum volume
appropriate for each pipetman is typically ten percent of the maximum. The dot
on the plunger is also color-coded and matches the color of the disposable tips
used with that pipetman. The table below shows the volume ranges, expected
accuracies, and the appropriate tips for each of the pipetmen you will be using.
In most experiment, accuracy is important when transferring small
volumes of liquid with a pipetman. A researcher needs to be sure that she is
transferring the volume desired with a reasonable degree of accuracy. The
research can be confident of this, provided two conditions are satisfied. First, the
pipetman must have been calibrated and tested for accuracy (this is usually done
on a yearly or semi-yearly basis). Second, the research must be using the
pipetman properly.
Pipetman
P-1000
P-200
P-20
Volume Range
(µl)
100-1000
20-200
1-20
Accuracy
Disposable Tip
Color
±10µl
±1µl
±0.5µl
Blue
Yellow
Yellow
Exercise 1: Familiarization with the pipetman/How to draw up and eject
liquids
Pick up a P-20 and se the volume setting for 10µl (reading from the top to
the bottom it should be ‘100’). Push the plunger down and notice that there is a
point that the plunger becomes more resistant and requires more effort to push
further. This point is called the “first stop”. Notice that the plunger can be
pushed well beyond the first stop until it reaches the “second stop”. Be sure you
can feel the first stop point and notice how far the plunger travels before reaching
this point. Reset the pipetman to 1.0µl (‘010). Again push the plunger to the first
stop. You should notice that the plunger travels a much shorter distance before it
reaches the first stop than it did when the pipetman was set for 10µl.
1. To transfer solution using a pipetman, set the volume dial to the desired
volume by rotating the volume adjustment knob. Note: the volume
setting should never be adjusted above the maximum volume specified for
a particular pipetman. The maximum volume is the volume shown on the
plunger. The table below shows how the volume display looks for each
pipetman when it is set at its maximum volume.
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Pipetman
P-1000
P-200
P-20
Maximum
Setting
1
0
0
2
0
0
2
0
0
2. Seat a disposable tip on the pipetman by placing the end of the barrel into
a tip and pressing down firmly.
3. Push the plunger to the first stop and place the tip into the solution you are
transferring. Slowly release the plunger to draw the desired volume of
solution into the tip. Note: If you release the plunger too fast it will draw
an inaccurate volume and plash solution into the barrel. Getting solution
into the barrel can damage a micropipet.
4. Place the tip in the tube that is to receive the solution. Press the plunger
to the first stop to expel the solution and then press to the second stop to
blow out any residual fluid.
5. Continue to hold the plunger down until you withdraw the tip from the tube
to avoid drawing the solution back into the tip.
6. Depending on the situation, the same tip may be sued again. In many
cases, however, it is necessary (and vitally important) to use a new tip for
each transfer. When you wish to eject the tip you are using, place the tip
over the appropriate waste container and press the tip ejector button. If
the tip is difficult to eject, it is likely that you are jamming the tips onto the
pipetman harder than necessary.
Exercise 2: Transferring small volumes with a pipetman
Obtain a 4 x 6-inch piece of blotting paper and a microcentrifuge tube
containing 2X loading dye. Following the protocol above, use a P-20 to spot the
following volumes of dye directly onto the paper in a linear order: 1µl, 3µl, 5µl,
7µl, 10µl, 15µl, and 20µl. Spot each volume twice to check your consistency.
Compare the spots you made with those of the instructor. If they don’t look right,
check with your instructor to determine the cause.
Exercise 3: Transferring volumes between tubes
This experiment simulates manipulations that would be performed when
mixing different volumes of several solutions. Real experiments using these
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techniques will be done during this lab and for the rest of the semester. These
include a bacterial transformation and preparation of bacterial plasmid DNA.
1. Obtain two microcentrifuge tubes and label them A and B.
2. Add the appropriate volumes of solutions I-IV to each tube following the
table below:
Tube
A
B
Solution I
100µl
150µl
Solution II
200µl
250µl
Solution III
150µl
350µl
Solution IV
550µl
250µl
3. You probably noticed that you have added a total of 1000µl to each tube. To
check that you pipeted accurately, set your P-1000 to 1000µl and withdraw the
solution mixture from the tube. Does the mixture just fill the tip? If there is
solution left in the tube or air in the end of the tip, there may be a problem with
your technique. If so, check with your instructor.
Exercise 4: Loading small volumes into a gel
On your bench you will find clear circular plates with a clear gel at the
bottom. These plates also have a small amount of buffer or water in them so
take care not to tilt the plate and pour out the liquid. If you look closely at the gel
you will also notice that there are several small slits (wells) in the gel arranged in
a straight line. Next week you will be loading real human DNA into the wells of a
gel that is connected to a power supply which will separate the DNA by size. We
will talk more about this later. For now we want you to practice putting a sample
into a gel.
1. On your bench you will find tubes labeled “G” for gel. These tubes have
30µl of dye and water in them.
2. Load the following amounts of liquid into the wells in sequential order: 5µl,
10µl, 15µl.
Tips: Draw up liquid by pushing to the first stop before inserting pipetman
into the liquid you want to draw up. Again, draw up the liquid into the tip
slowly to avoid any air bubbles in the tip. Any air bubbles can seriously affect
the loading of a gel. Use your other hand to steady the pipetman as you
insert the tip into the well of the gel. DO NOT jam the tip into the well as this
will puncture the well and your sample will leak out underneath the gel.
Instead, gently rest the tip against the side of well and slowly press the
plunger allowing the sample to be ejected into the well. DO NOT press the
plunger to the second stop (which means that you will still see some sample
remaining in your tip) because this forces air into the well which can cause
the sample already in the gel to be blown out.
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Spooling Purified DNA
INTRODUCTION
Each of the 46 chromosomes in one of your cells contains one DNA molecule
that is an inch or two long, though it is far too slender to be seen with any but the
most powerful electron microscopes. If we could enlarge one of these
DNAmolecules enough so that we could see it — let’s say so that it was about
the same diameter as one of the hairs on your head — we would find that it was
several miles long!
In this exercise, we will take advantage of this long, thin, “threadlike” shape of
DNA molecules to “spool” them, which is to say, we will wind them up on a
wooden stick like a piece of thread. As you probably know, cotton fibers (which
are individually a couple of inches long and quite thin) can be combined into one
long, continuous thread, because they tend to stick to one another and line up
side by side. The same thing can happen when DNA molecules come out of
solution — if we pull on them from one end. We will do this by slowly twisting a
stick in the region where DNA is beginning to precipitate. Each DNA molecule
that is initially caught and wound around the stick will catch and pull on several
other molecules, each of which will then catch and pull on others. Thus, if we
work carefully, we can wind all of the DNAmolecules in a test tube into one long,
continuous thread. Although DNAmolecules are so thin that you couldn’t
possibly see one of them with your naked eyes, if you wind up many such
molecules together (as you will in this exercise), the DNA becomes visible, and
its properties can be studied.
MATERIALS
For each group of four students:
1 test tube of DNA
1 test tube of alcohol
1 wooden stick
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PROCEDURE
1. Observe the alcohol and DNA solutions in the test tubes. Can you tell which
one is the DNA? How?
2. Uncap the tubes and hold the tube that contains the DNA at an angle.
3. Carefully transfer the alcohol from its tube into the DNA tube. Pour very
slowly, so that the alcohol does not mix with the DNA solution and stir it up.
4. Gently insert the wooden stick through the alcohol layer to the interface where
the two liquids meet. Twirl the stick gently.
5. Now slowly lift the stick from the tube and observe the material clinging to it.
How long a fiber can you pull from the tube?
10
Models of DNA Structure
Stick Model
Use the DNA models provided to answer the questions in your lab report (next
page).
Computer Tutorial
Turn on your computers and open the Bio 280 home page:
http://www.nslc.wustl.edu/courses/Bio280/bio280.html
Open the ‘course contents’ section using the password. Click on the site DNA
Structure by Eric Martz. Open item #1, Double helix by element: base pairs,
hydrogen bonding. Click on the boxes at the bottom of the screen to highlight the
Backbone, Bases, and H-bonds. Clicking on either the X Spin or the Y Spin box
enables you to spin the molecule for different perspectives. (Remember to Reset
the image after each manipulation.) Doing a Y spin in the Thick mode is a good
way to see that the sugar-phosphate backbone is on the outside, the base pairs
on the inside. Doing a Y spin in the Space fill mode (done by clicking on the both
the Y Spin and the Space fill boxes) is a good way to see the major and minor
grooves. Using the Y spin, follow the position of the major and minor grooves
and check strand polarity.
Use this tutorial to answer the questions in your lab report.
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12
Bio 280 Lab Write-up: DNA Models
Name____________
Date of Investigation: / /
Investigation 1: DNA stick model
In groups of two or three examine the DNA model on your bench. As you answer
the following questions you should gain a better familiarity with the structure of
DNA.
1. By comparing the DNA model before you with text-book structures,
determine which colors on the model represent which atoms.
Black=______Red=_______White=_____Yellow=_____ Blue=_____
2. What are two quick and easy ways to distinguish A-T and G-C base pairs
on this model?
3. How can purine and pyrimidine members of a base pair be distinguished?
4. Using a metric ruler, measure the distance (in cm) from the middle of one
base pair to the middle of the next.
distance = ________ cm
a.) What is the scale of this model; i.e. how many angstroms are
represented by one centimeter on the model?
b.) What is the magnification factor; i.e. how many times bigger is this
model compared to a real DNA molecule?
5. The average gene codes for a polypeptide approximately 333 amino acids
long. Knowing that it takes 3 base pairs worth of DNA to code for each
amino acid, what fraction of a typical gene is represented by this model?
6. If a typical gene’s worth of DNA was constructed on the scale of this
model, approximately how many meters high would the model stand?
7. How many base pairs are there per revolution of this double helix?
Calculate the degrees of rotation between adjacent base pairs in DNA.
13
8. Examine the deoxyribose units associated with a single base pair in the
model. How do these sugars reveal the antiparallel nature of the sugarphosphate backbones?
9. What is the A+T/G+C ratio for this model? What is the A+G/C+T ratio?
Why are they different?
Investigation 2: Computer-based models
After reviewing the basic B-form DNA structure, select an A-T base pair (click on
the AT box). Moving the mouse while depressing the shift key allows you to
adjust the size of the base pair to the field of view. Rotate the base pair (by
moving your mouse) to achieve an ‘edge-on’ view of one ring structure at a time.
1. Are the purine and pyrimidine rings planar? Is the sugar ring planar?
2. Repeat your observations for the G-C base pair (by clicking on the GC
box). Are the purine and pyrimidine rings planar?Is the sugar ring planar?
Click Back on the tool bar to reach the DNA Structure (by Eric Martz) page.
Click on item #4, Ends, Antiparallelism. Examine the ends of each strand.
Identify the deoxyribose sugar backbone of each strand. Look at the
deoxyribose sugar rings at the end of each strand.
14
PCR and Genetic Variation in Humans
Alu Part 1
Adapted from Cold Spring Harbor Laboratory DNA Learning Center by the Washington University
Science Outreach Program
Background:
Since its development two decades ago, polymerase chain reaction
(PCR) has become a tool used almost universally by geneticists. PCR is used to
quickly amplify, or create millions of copies of, specific regions of a particular
segment of a DNA strand. PCR allows researchers to study very small amounts
of DNA without resorting to laborious cloning procedures. The technique has
had an impact in many areas of biology and has greatly facilitated the field of
forensics.
Although the DNA from different individuals is more alike than different,
there are many regions of the human chromosomes that exhibit a great deal of
diversity. Such variable sequences are termed polymorphic, meaning many
forms. These polymorphic sequences provide the basis for genetic disease
diagnosis, forensic identification, and paternity testing.
In this lab exercise, PCR will be used to amplify a nucleotide sequence
from human chromosome 8 to look for an insertion of a short DNA sequence
called Alu, within the tissue plasminogen activator (TPA) gene. Alu sequences
are thought to be derived from the 7SL RNA gene. This gene encodes for the
RNA component of the signal recognition particle which functions in protein
synthesis. Alu elements are approximately 300-bp in length and derive their
name from a single recognition site for the endonuclease Alu I, located near the
middle of the Alu sequence.
An estimated 500-2000 Alu elements are thought to exist, and most of
them are found in the human genome. A few have appeared relatively recently,
within the last one million years, and are not present in all individuals. One such
Alu element, called TPA-25, is found within an intron of the TPA gene. This
insertion is dimorphic, meaning that it is present in some individuals and not in
others. PCR can be used to screen individuals for the presence, or absence, of
the TPA-25 insertion.
In this experiment, oligonucleotide primers flanking the insertion site will
be used to amplify a segment of the TPA gene. The expected product of the
PCR reaction will be a 400-bp fragment when TPA-25 is present and a 100-bp
fragment when it is absent. Each of the three possible genotypes –
homozygous for presence of TPA-25 (400-bp fragment only), homozygous for
the absence of TPA-25 (100-bp fragment only), and heterozygous (400-bp and
100-bp fragments) – will be able to be determined following electrophoresis of
our PCR reaction products in an agarose gel.
15
Description of Lab Exercise
Our source of template DNA will be a sample of several thousand cells
obtained from inside your mouth. The cells will be suspended in a solution
containing Chelex, a resin that binds metal ions (and that can also inhibit a PCR
reaction). The cells will be lysed, or broken open, by boiling and then
centrifuged to remove cell debris. A sample of supernatant containing your
genomic DNA will be mixed with Taq DNA polymerase, oligonucleotide
primers, the four deoxynucleotides (A, T, C, G), and the cofactor MgCl2.
Temperature cycling will be used to denature the template DNA, anneal the
primers, and extend a complementary DNA strand. The size of the amplification
product(s) will depend upon the presence or absence of the Alu insertion at the
TPA-25 locus on each copy of chromosome 8.
We will compare the genotypes of different individuals by loading aliquots
of your amplified samples into wells of an agarose gel. The lab instructor will
load DNA size markers into other wells. The bands of the markers will be used
to estimate the sizes of your PCR products. Following electrophoresis,
amplification products appear as distinct bands in the gel – the distance a DNA
fragment travels from the starting well is inversely proportional to its molecular
size. The larger TPA product (containing the 300-bp Alu element) will not
migrate as far from the well as the smaller TPA product (missing the 300-bp Alu
element). In a successful experiment, one or two amplified bands will be
observed, indicating that you are either homozygous or heterozygous for the
TPA-25 locus.
Experimental Protocol
NOTE: PCR is extremely sensitive to contamination. A few foreign cells in your
preparation can ruin your results. For this reason, it is extremely important to
maintain sterility at all times while conducting the following procedures.
A. Cell Extraction and DNA Isolation
1. Put on safety glasses and latex gloves.
2. Find one 1.5 mL microcentrifuge tube containing 10% Chelex
(pronounced KEE-lex) and use a permanent marker to label the top or
side with your 3 letter initials. (Note that Chelex is not actually a solution,
but a slurry of resin coated beads in water. You will see the beads settled
at the bottom of the tube.)
3. Your instructor will pass around a glass tube containing flat wooden sticks.
These sticks have been autoclaved and are sterile. Your instructor will
demonstrate how to remove a stick and keep it sterile. Open the glass
tube and gently tap out a single wooden stick.
4. With the sterile end of your wooden stick, thoroughly scrape the inside of
your cheeks.
16
5. Open your Chelex tube and put the end of your wooden stick (with cheek
cells) into the tube. Gently twirl or stir your stick to dislodge the cells into
the tube. Close the Chelex tube. Discard your wooden stick in the trash.
6. Finger vortex the Chelex tube for 10 seconds.
7. Place your 1.5 mL microcentrifuge tube in a floating rack in the boiling
water bath for 10 minutes.
8. Place your boiled sample on ice for 2 minutes.
9. Centrifuge the sample for 30 seconds to pack the Chelex beads to the
bottom of the tube. Your extracted DNA will remain in the supernatant
above the beads.
B. PCR Amplification
1. Put on safety glasses and latex gloves. Use a permanent marker to label
the top of a sterile 0.6 mL microcentrifuge tube with your initials. This is
your PCR reaction tube.
2. Using a P-20 micropipettor, pipet 10 µL of your extracted DNA sample into
the reaction tube. Be careful to pipet only the supernatant of your DNA
sample – avoid the Chelex beads. Any Chelex in the PCR reaction will
inhibit the enzyme and ruin the reaction.
3. In the ice bucket on your bench you will find a tube labeled Amp Mix. Use
a P-200 micropipettor to pipet 30 µL of this amplification mixture into your
0.6 mL PCR reaction tube. This mixture contains oligonucleotide primers,
the four deoxynucleotides (A, T, C, G), Taq polymerase, and buffers.
4. Use a P-20 micropipettor to pipet 10 µL of MgCl2 solution to your 0.6 mL
PCR reaction tube.
5. Your DNA is now ready for amplification in the PCR machine, or thermal
cycler. Your instructor will collect your reaction tube, place it in the PCR
machine, and start the program. The reaction will proceed as follows:
1 cycle
94˚C for 5 minutes
30 cycles
94˚C for 1 minute
58˚C for 2 minutes
72˚C for 2 minutes
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18
Biology 280 Lab Write-up: Alu
Name:________________________
Date of Investigation:
Below is the sequence of human DNA called the TPA-25 locus that we are
amplifying in class using Polymerase Chain Reaction or PCR. The sequence
below also contains the Alu insertion. The sequence shows spaces every ten
bases.
5’ CACATATTGT
TTAAGGGTCC
CTTTATTGCA
TGTCTGACAA
ACGCCTGTAA
TCAGGAGATC
AAAACTACAA
TACTTGGGAG
TGCAGTGAGC
AGACTCCGTC
ACTGGGTAAC
AAGGTCAAGA
GGTTGACTGA
ATGGGATTGA
TAATCCCAGC
GAAAAGCAAG
TGGCCTGTAA
ATGATTTGTA
CCCTATGAGA
TCCCAGCACT
GAGACCATCC
AAAATAGCCG
GCTGAGGCAG
CGAGATCCCG
TCAAAAAAAA
AAAGTTAAAG
TGAACTCGGT
GTCTCCTTCT
AAATCTTAAA
ACTTTGGGAG
GTCTACCAGT
CCATTTAGTC
AGAGTTCCGT
TTAGAACACT
TTGGGAGGCC
CGGCTAAAAC
GGCGTAGTGG
GAGAATGGCA
CCACTGCACT
AAAAAAAAAA
AGAAGTTCTC
GTCCTCCCTC
ACTCTTACAT
CTCCTGGCCT
–3’
TTTCCAACCT
CTCAGCTGTT
AACAGGACAG
ACGGCCGGGC
GAGGCGGGCG
GGTGAAACCC
CGGGCGCCTG
TGAACCCGGG
CCAGCCTGGG
AAAAGAACAC
CTAGGGTGGG
CCAGCTCAGT
GGCCTGTGAT
GGTGTGGTGG
AAATCCCAAG
CTCCTGACAT
CTCACAGTTC
GCGGTGGCTC
GATCACGAGG
CGTCTCTACT
TAGTCCTGGC
AGGCGGAGCT
CAACAGAGCG
TACATTACTG
GGTGTGCTGC
GGTTTTCATT
GTGGCTGAAA
TGCATGCCTG
The sequence of the primers that we used in this experiment are shown below:
Primer 1: 5’-GTAAGAGTTCCGTAACAGGACAGCT-3’ (upstream primer)
Primer 2: 5’-CCCCACCCTAGGAGAACTTCTCTTT-3’ (downstream primer)
1. Draw a line under the sequence in the TPA gene where each primer binds
with the proper label. Remember: The Alu sequence above shows only one
strand of the double-stranded gene fragment. Since the downstream primer
actually binds to the complementary strand, you will need to determine either
the complementary strand for the gene or for the primer in order to determine
where it binds.
19
2. Now underline the region in the sequence shown that represents the Alu
insertion and label the line.
3. If you were interested in amplifying only the region from nucleotide 65 to
nucleotide 400, what 20 base pair primers would you use? Remember to
label the 5’ and 3’ ends of each primer. (Assume the first nucleotide shown is
nucleotide 1.)
3. As you know, humans are diploid, meaning they have two copies of every
chromosome. It is possible then that some individuals have one
chromosome with the insertion and one chromosome without. What
would you expect to see if the DNA from such an individual was amplified
and run on an agarose gel? (Draw a picture of the gel with labels.)
Answer questions 4 – 7 after Part 2 of the Alu lab (running the gel).
4. What is your Alu genotype?
5. What is the distrubution of genotypes for the class?
20
6. If a crime had been committed, could you identify the perpetrator from a
DNA sample using the Alu locus? (Assume that a DNA sample has been
voluntarily provided by all suspects in the case.) Explain your reasoning.
21
22
PCR and Genetic Variation in Humans
Alu Part 2
Adapted from Cold Spring Harbor Laboratory DNA Learning Center by the Washington University
Science Outreach Program
Background: This week you will run your amplified DNA on an agarose gel to
separate alleles. If your amplification was successful, you will be able to
determine your D1S80 genotype. We will then quantify occurrences of the
alleles in our class.
Day 2:
A. Running an agarose gel
1. Your instructor will return your PCR reaction tube to you. Your reaction
tube now contains your PCR product.
2. Put on safety glasses and latex gloves. Find a tube of Orange G loading
dye. Use a P-20 micropipettor to add 10 µL of loading dye to your PCR
reaction tube.
3. Use a P-200 micropipettor to load the entire 60 µL into one well of the
2.0% agarose gel. Record your name on the gel diagram sheet next to
the appropriate well number for your PCR product. This is so you will
know which lane of the gel contains your PCR product.
4. The gel should run for ~1.5 hours at 100V.
B. Analyzing the gel
When the electrophoresis is finished, your instructor will photograph the gels
so the results can be visualized and analyzed. Be sure to note the number and
size of any bands in your lane of the gel.
23
24
Sterile techniques and transforming bacteria
with DsRed.
Adapted from M.Grupe ‘What color is your colony?’ teacher manual, Washington University
Science Outreach 2005
A. Practicing Microbiological Techniques
Background: In this laboratory, we will be using the bacteria E. coli. To avoid
contamination of your experiment with unwanted bacteria, it is necessary to use
sterile techniques and work with all reagents carefully. Wipe your work area with
bleach before and after your laboratory and dispose of used pipette tips, tubes,
etc. in the containers provided by your instructor. Always wash your hands
thoroughly after each laboratory exercise.
1. Put on your safety goggles. Observe your plate of bacteria. To avoid
contamination, do not open the lid yet. Notice that the bacteria grow in
clumps called colonies. Each colony started as one bacterium that then
multiplied many times. All of the bacteria in a colony usually are identical.
Bacteria grow and divide quite rapidly. Under the best conditions, E. coli
cells can divide every 20 minutes or so.
2. Transfer a colony of bacteria into the tube of nutrient broth. Follow these
steps:
a.
b.
c.
d.
Carefully remove an inoculating loop from the package. Do not
touch the loop to anything.
Carefully tilt the lid of the petri dish containing the bacteria, without
moving the lid away from the dish. This protects the agar from
contamination with organisms floating in the air.
Insert the loop end of your inoculating loop and touch a single
colony of the bacteria with the tip. Withdraw the loop and replace
the lid.
Place the loop containing the bacteria into the tube of nutrient
broth. Twist the loop to mix the E. coli with the broth. Check to be
certain the bacteria have come off the loop. Remove the loop.
Recap the tube. Dispose of the loop in the bleach solution provided
at your lab station.
3. Next, you will practice transferring a bacterial suspension from a tube to a
petri dish.
a.
b.
Label the bottom of a petri dish containing sterile nutrient agar with
your name and the date.
Set the micropipettor to 100 µl. Using a sterile large tip, transfer
from the tube 100 µl of the bacterial suspension that you just made
25
c.
onto the agar in the petri dish. Dispose of the tip in the waste
container.
Using a new sterile loop, spread the broth containing the bacteria
over the surface of the agar. Make a zigzag motion across the agar.
Turn the plate 90˚ and repeat the zigzag motion. Dispose of the
loop in the waste container. Put the cover on the plate and seal it
with a piece of plastic wrap. Return your plate to your instructor.
B. Transforming Bacteria: Part 1
1. Get a cup with ice in it. Locate the tube labeled “W.” This tube contains a
very small amount of water. Put your initials on the tube. Gently tap the
tube on the counter to shake the drop of water to the bottom of the tube.
This tube will be your control tube. Place it in your cup of ice.
2. Locate the tube labeled “DNA.” This tube contains the DNA with the genes
that will change a bacterial colony color. Put your initials on the tube.
Place it in your cup of ice.
3. Locate the tube labeled “C.” It contain a chemical called calcium chloride,
CaCl2. Calcium chloride makes bacteria “leaky,” so that big molecules like
DNA can get inside the cells. Put your initials on the tube and place it in
the ice.
4. Locate the petri dish culture of E. coli. (You may be sharing this with one
or more groups.)
5. Use the inoculating loop to transfer a mass of bacteria to the tube labeled
“C.” Then use the micropipettor to mix the bacteria with the liquid in tube
“C.” To do this:
a.
b.
c.
d.
e.
f.
fluid in
g.
Tilt the lid of the petri dish, holding it over the dish.
Pick a colony off the surface of the agar with the loop, then close
the dish
Open tube “C,” put the loop with bacteria into the liquid, and gently
spin the handle of the inoculating loop to knock off the clump of
bacteria. Be sure the bacteria came off the loop! Dispose of the
loop.
Set your micropipettor to 250 µl.
Place a sterile tip on the micropipettor.
Pick up the tube that now holds the bacteria and gently pipette the
and out to break up the clump of bacteria. Cap the tube and
dispose of the tip.
Put the tube with the bacterial mixture into the ice.
26
6. Add 250 µl of the bacterial suspension you just made to the tube
containing water.
a.
b.
c.
d
e.
Set your micropipettor to 250 µl.
Place a sterile tip on it.
Transfer 250 µl of the bacterial suspension from tube “C” to tube
“W.” Pipette in and out several times to mix.
Place tube “W” on ice.
Dispose of the tip.
7. Repeat the steps in 7 to add 250 µl of the fluid in tube “C” to tube “DNA”.
8. Start timing both tubes (“W” and “DNA”) at this point. Leave them on ice
for 15 minutes.
9. At the end of 15 minutes, bring the tubes (still on ice) to the water bath.
Check the temperature in the water bath to make sure it is 42˚C. Transfer
the two tubes from the ice to the water bath. Leave the two tubes in the
water bath for exactly 90 seconds.
10. Remove the tubes from the water bath and put them back on ice for one
minute. After that, they can be at room temperature.
11. Locate the two petri dishes, one labeled “water,” the other labeled “DNA.”
Put your group name and hour on the bottom of each dish.
12. Set the micropipettor at 250 µl. Place a new tip on the micropipettor and
transfer 250 µl from tube “W” onto the agar in the plate dish marked
“WATER”. Dispose of the tip in the waste container.
13. Using a sterile loop, spread the bacteria across the surface of the agar.
Remember to zigzag, then rotate the dish and repeat. Dispose of the loop.
14. Place a new tip on the micropipettor and transfer 250 µl from tube DNA
onto the plate marked DNA.
15. Using a sterile loop, spread the bacteria across the surface of the agar.
Remember to zigzag, then rotate the dish and repeat. Dispose of the loop.
Seal both dishes with plastic wrap and place them where your instructor
directs. Incubate at room temperature.
D. Viewing Plates
Observe both of your agar plates and record what you see in the space below.
Be sure to record when bacterial colonies can first be seen on each plate.
Meanwhile, begin filling out the laboratory write up on the next two pages.
27
28
Biology 280 Lab Write-up: Sterile Techniques and Shine On
Name:________________________
Date of Investigation:
1. In this laboratory we used molecular cloning techniques to isolate and
amplify DNA fragments. How is DNA amplified in a cloning experiment?
2. How is this amplification different from a PCR experiment?
3. In a cloning experiment like this, what does it mean to say that a bacterial
cell has been transformed?
4. How do we separate transformed cells from untransformed cells? Be
specific.
5. What are the controls in this experiment? Why are they important?
6. What is the major trait we are trying to change in the bacteria? Extra point
if you name both traits changed in this experiment
29
7. What is it that causes the bacteria to change color?
8. Does it matter which organism the piece of DNA we insert into the bacteria
came from? Why or why not?
30
Learning to Clone DNA
Part One: Construct Creation and Bacterial
Transformation
Background: Molecular cloning is a process by which DNA fragments are
isolated and amplified. This technology is based on the ability to cut DNA
fragments precisely (using restriction enzymes), join DNA fragments together,
and make many copies of the new DNA. In this lab we will cut up yeast DNA
(our source DNA) and join these DNA fragments (using a DNA ligation reaction)
with a circular piece of DNA that makes copies of itself naturally when inserted
into a bacterial cell. The circular piece of DNA is called an autonomously
replicating vector or plasmid molecule. In this lab you will use a small (~3kb)
double-stranded DNA vector called pBlueScript (pBS; see map below). Like all
good vectors, pBS contains the necessary information to support its autonomous
replication in a host (E.coli bacteria in this case). This plasmid is engineered to
allow it’s host cell to grow in the presence of an antibiotic (ampicillin in this case).
This trait will allow us to easily identify which cells contain our plamid. This
plasmid also contains a region with “unique” cutting sites. These are called
unique sites because they occur only once in the vector and provide useful
positions to insert DNA fragments from the source organisms.
For this experiment, you will be given a sample of pBS that has been cut once
with the restriction endonuclease EcoR I to generate 5’- P AATTC
G overhangs.
The cut plasmid was subsequently treated with a phosphatase (an enzyme that
cuts off phosphates), yielding a DNA fragment that looked like this:
5’- OH AATTC
Now this cut in the circular piece of DNA will not seal back together, a process
called vector re-ligation, but remain open to receive our fragment of source DNA.
A DNA ligase reaction requires a 5’-phosphate and a 3’-hydroxyl as substrates.
The source insert DNA in this lab will be from the yeast Saccharocyces
31
cerevisiae. You will be given a sample of S. cerevisiae DNA that has been cut
with the restriction enzyme EcoR I but NOT treated with phosphatase.
pBlueScript (2.96 kb)
ampicillin
resistance
marker
Multiple
cloning
site
EcoRI
DNA
replication
origin
The source and vector DNA samples will be ligated in a small reaction
containing buffer, ATP, and DNA ligase. As a negative control, each group will
set up a ligation omitting the source DNA.
After a brief room temperature incubation, the ligation products will be mixed
with E. coli cells that have been pretreated with CaCl2 to make their cell
membranes porous, thereby facilitating uptake of DNA molecules from the
external media (cells prepared in this manner are known as ‘competent’ cells).
The cell + DNA mixture will be ‘heat shocked’ to stimulate DNA uptake (by a
poorly understood mechanism). The cells will then be allowed to recover from
this harsh treatment in fresh non-selective media. Finally, the cell mixture will be
spread on selective LB + ampicillin plates allowing the bacteria that have taken
up plasmid DNA to replicate while killing the bacteria that failed to take up a
plasmid. The result will be the growth of colonies, each colony representing an
independent ‘transformation’ event. All bacteria in a colony are clones (i.e.,
genetically identical individuals derived from a single precursor) that harbor the
same plasmid + insert combination.
The actual molecular cloning experiment that you begin today will be
continued over the next four weeks.
Week One:
You will perform a DNA ligation reaction with prepared bacterial plasmid
vector and source (in this case, yeast genomic) DNA fragments. The
32
products of the ligation reactions will then be mixed with permeabilized
(competent) E. coli host cells. Bacterial cells that take up plasmid vector DNA
(+/- inserts) will be selected on antibiotic nutrient plates to recover individual
clones. The day after lab, you will examine the transformation plates, count
colonies and start a liquid bacterial culture using a clone that potentially
contains an insert as an inoculum.
Week Two:
You will prepare plasmid DNA from your bacterial culture. Using this DNA,
you will set up a restriction endonuclease digestion of your potential plasmid
clone. You will also set up a sequencing reaction that will yield the sequence
of your inserted yeast DNA fragment, if one is present.
Week Three:
You will analyze the results obtained from your restriction digestions and the
results obtained from your DNA sequencing reactions.
Experimental Protocol: (Work in groups of 2)
Enz mix
(buffer + DNA ligase)
Vector DNA
Source DNA
dH20
ATP [5 mM]
+ insert ligation
– insert ligation
4 µl
2 µl
2 µl
0 µl
2 µl
10 µl
4 µl
2 µl
0 µl
2 µl
2 µl
10 µl
Ligation:
1. Find two tubes labeled E and label them with your initials. Label one tube as
+ (for ‘plus insert DNA’ ligation) and the other tube as – (for ‘minus insert
DNA’ ligation).
2. To each ‘Enz’ tube add 2 µl of vector DNA from tube labeled V (REMEMBER
TO CHANGE YOUR PIPET TIP AFTER EACH TRANSFER!!!)
3. To the ‘+’ tube add 2 µl of source DNA. For the control ligation, add 2 µl of
dH20 to the ‘–’ tube.
4. Add 2 µl of ATP to each of the ligation tubes to start the reaction.
*The reaction mixture set-up in steps 1-4 is summarized in the table above.
33
5. Tap the tube to mix the reaction and spin the tube briefly in the
microcentrifuge to collect the liquid in the bottom of the tube. Incubate the
reactions at room temperature for ≥15 minutes.
6. After room temperature incubation, place each ligation tube onto ice for 5
minutes to cool.
Transformation
7. To each tube, add 100 µl of competent cells (always keep the cells cold and
use sterile technique!). Quickly mix by gently tapping the tube and place the
cells + DNA back on ice to incubate for 20 minutes.
8. ‘Heat shock’ the cells by placing the tubes in a 37˚C waterbath for 2 minutes.
9. To each tube, add 500 µl of LB liquid broth. Mix tube by tapping. Incubate at
37˚C for 10 minutes.
10. Remove 100 µl of liquid from each tube and spread on the surface of an LB +
ampicillin plate. Incubate at 37˚C overnight.
* In parallel, your lab instructor will carry out three additional control
experiments. Competent cells that were not mixed with DNA (‘Cells only’)
will be plated on 1) LB without antibiotic (a positive control for cell viability),
and 2) LB + ampicillin (a control to confirm the efficiency of the antibiotic
selection). In addition, competent cells mixed with unligated vector + insert
DNA will be plated on LB + ampicillin as a negative control to determine the
number of colonies that result from residual uncut vector DNA.
11)On the following day between Noon and 5 PM: Retrieve your plates and
the additional control plates from the incubator in Rebstock 132. Count the
colonies on each plate and record below:
Plate:
Cells only
No amp
Cells only
plus amp
vector +
insert
unligated
–insert
ligation
+ insert
ligation
Colony #
After you record your data, pick ONE well-separated colony from the +
ligation plate and inoculate an LB + ampicillin liquid culture. A workbench and
sterile wooden sticks will be available in Rebstock 132 to start your cultures.
34
Remember to use sterile technique. Both you and your lab partner should
each inoculate a culture. Place your inoculated tubes in the rack designated
for your section. At the end of the day, your cultures will be incubated
overnight at 37˚C and saved at 4˚C until next week at which time you will
prepare plasmid DNA from your culture. Be sure to label your tube with your
name and lab section!
35
36
Bio 297 Lab Write-up: Learning to clone DNA.
Name___________________
Date of Investigation :
Answer the following questions after Part 1:
In the table below, indicate your results for this experiment. Count the number
of colonies on each plate and record them. If there are more than 500 colonies
just record ‘>500’; if there is a lawn record ‘lawn’.
Plate
Reaction contained:
1
Cells only
Ampicillin
in plate
no
2
Cells only
yes
3
Cells + vector, + insert,
no ligase
Cells + vector, no
insert,
+ ligase
Cells + vector, + insert,
+ ligase
yes
4
5
Your results
yes
yes
1. If this experiment worked correctly, you will see colonies on Plate #5.
Explain the presence of bacterial growth that you see on plates #1 through
4. Based on the presence or absence of bacterial growth on plates 1-4,
can you say that your experiment was successful or not? Explain.
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
______________________________________________________________
_________________________________________________
2. Using your notes from class and any outside materials you need, make a
diagram illustrating the first three initial phases of our cloning experiment.
a. Ligation phase (Draw everything you know about vector and insert.
How are they cut, phosphatated, and ligated?)
b. Transformation
c. Plating
37
Answer the following questions after Part 2:
1. Using your notes from class and any outside materials you need, draw the
DNA isolation experiment in two steps. 1) What did the cell look like
before the plasmid was isolated? 2) What was the result after the
plasmid isolation (miniprep experiment)?
2. Using your notes from class and any outside materials you need, draw a
picture of the following:
a. The vector cut with EcoRI, no insert.
b. The vector cut uncut (3 possibilities)
c. The vector cut with EcoRI, 1 insert.
38
Learning to Clone DNA
Part Two: Clone Characterization
Background:
Last week you picked a single colony off of your ‘+ insert ligation’ plate and
inoculated a liquid culture. Each colony should represent a single clone. In this
case, the clone is a particular DNA molecule. If the experiment was successful,
the clone should be a chimeric molecule comprised of the pBS vector and at
least one of the ~3 X 103 EcoR I fragments present in the source yeast genome.
This week, you will isolate plasmid DNA from your inoculated culture and cut the
DNA with the restriction enzyme EcoR I. The aim of this exercise is to determine
the structure of the clone, enabling you to answer the following questions: 1) do
you have a single insert, multiple inserts or no insert? 2) can the insert(s) be ‘cut
out’ of the vector with EcoR I as expected? 3) what is the size of the inserted
fragment or fragments?
To purify your cloned DNA, you will use a standard ‘alkaline lysis’ bacterial
plasmid miniprep procedure that allows isolation of plasmid DNA free of the
bacterial chromosomal DNA. The protocol involves five steps. First, the
bacterial cells are harvested by centrifugation. Second, the cells are
resuspended in a buffered solution (#1) and lysed by the addition of Solution #2
containing NaOH and sodium dodecyl sulfate (SDS, a common detergent.
Check the fine print on most shampoo bottles.). The NaOH also serves to
denature double-stranded chromosomal and plasmid DNA. The third step is the
neutralization (with Solution #3) of the alkaline lysis solution to allow doublestranded DNA to reform. The strands from each small circular plasmid DNA will
remain interlocked and will quickly reanneal to reform a double-stranded plasmid.
However, the complementary strands of the larger linear chromosomal DNA
fragments will not reanneal properly and will become irreversibly denatured and
insoluble. In addition, the potassium ions in the neutralization solution (#3) cause
the detergent to precipitate with insoluble denatured proteins and improperly reassociated chromosomal DNA, carbohydrates and cellular debris. This
unwanted material is pelleted by centrifugation in the fourth step, leaving a
supernatant containing the desired plasmid DNA. The fifth step is the collection
of the plasmid DNA by ethanol precipitation and centrifugation. The plasmid
DNA pellet will be resuspended in a dilute buffer (TE) plus RNase to degrade
unwanted RNA.
After isolating plasmid DNA from your bacteria, you will set up a restriction
endonuclease digestion. A portion of your newly isolated plasmid DNA will be
added to a tube containing a mixture of EcoR I and buffer. Your restriction
digests will be incubated for several hours at 37˚C and the resulting fragments
size-fractionated by agarose gel electrophoresis. Next week in lab, you will
analyze the data from your section.
39
In the final step of the lab today, you will set up a reaction that will enable
sequencing of your inserted DNA fragment. The sequencing protocol we will
employ makes use of a technique with which you should already be familiar:
PCR. Your plasmid + insert DNA will be used as a template to synthesize
additional copies of this DNA. As in a typical PCR reaction, the DNA polymerase
being used is stable at high temperatures and allows copies of your template to
be generated after ‘melting’ a double stranded sample into single strands. Both
deoxynucleotide triphosphates and dideoxynucleotide triphosphates will be
included along with a DNA polymerase, your DNA template and a primer
(complementary to your plasmid DNA, usually to a region within the multiple
cloning site). As you remember from lecture, dideoxynucleotide triphosphates
are missing a 3’-OH group. Without this 3’-OH group, another nucleotide cannot
be added to the lengthening DNA chain; therefore, polymerization of the new
DNA strand stops when one of these nucleotides is used. The ratios of dNTPs to
ddNTPs has been formulated so that polymerization is terminated by
incorporation of a ddNTP at least once at each base, resulting in the production
of many DNA strands, each of which is of unique length, differing in size by one
base. The differently-sized products that are generated by this sequencing
protocol can be separated by gel electrophoresis. Each dideoxynucleotide is
‘labeled’ with a colored dye specific to the nucleotide (for example,
dideoxyadenosine triphosphate is labeled with a green dye while ddGTP is
labeled blue), and the color of each piece of DNA is detected and recorded as it’s
run off the gel. A computer analyzes this color data and produces a print out of
your DNA sequence. Of course, the actual reading of the sequence will not be
done by you; this process is all automated. All you need to do is set up the
reaction, put it in the PCR machine, and analyze your results!
Experimental protocol: (Work in pairs)
DNA isolation
1. Transfer 1000 µl of your overnight culture to a clean microcentrifuge tube.
2. Pellet cells by centrifugation for 1 minute.
3. Remove the supernatant using a P-1000 pipetman. Make sure there is no
excess liquid remaining on the cell pellet.
4. Add 250 µl of Solution #1 to the cell pellet.
5. Vortex the tube to resuspend the cell pellet. Make sure that no pellet remains
visible on the bottom of the tube.
6. Add 250 µl of Solution #2. Close the cap and tip the tube back and forth to
mix (Do not vortex!). Let the tube sit at room temperature for one minute.
The mixture should become clear (loss of turbidity due to cells lysis) and
viscous (long DNA polymers spill out of the broken cells).
40
7. Add 350 µl of Solution #3. Close the cap and tip the tube back and forth
again to mix (Do not vortex here either). You should see a yellow-white
precipitate form. Continue tipping back and forth for 30 seconds.
8. Spin the tube in a high speed microcentrifuge (on the side bench) for 10
minutes to pellet the debris (including chromosomal DNA, proteins,
carbohydrates).
9. Carefully remove the entire volume of the supernatant and transfer to a clean
spin column / microcentrifuge tube. Be careful not to trasfer any of the pellet
or floating particles. Discard the tube with the precipitate in the biohazard
waste.
10. Spin the DNA-binding spin column / microcentrifuge tube in the
microcentrifuge on your bench for one minute. All of the liquid will be forced
through the column and any DNA in the liquid will bind to the resin on the
column. Discard the liquid in the microcentrifuge tube and put the column
back into the same tube.
11. Transfer 700 µl of Solution #4 onto the column. Spin the tube in the
microcentrifuge on your bench for one minute. This step serves to wash any
non-specifically bound material off of the column. The plasmid DNA remains
bound to the column during this was step.
12. Discard the liquid in the microcentrifuge tube and replace the column back in
the same tube.
13. Spin the column / microcentrifuge tube (empty) in the high speed
microcentrifuge (on the side bench) for one minute. This spin results in the
removal of any residual liquid from the column. The plasmid DNA remains
bound to the column.
14. Discard the microcentrifuge tube and place the column (still with your plasmid
DNA) in a new, labeled microcentrifuge tube.
15. Elute (wash off) your plasmid DNA from the column by pipeting 35 µl of dH2O
onto the column. Spin the column / microcentrifuge tube in the
microcentrifuge on your bench for one minute. Your plasmid DNA is now in
the liquid in the microcentrifuge tube; the column can be discarded. Use this
DNA to set up both the restriction digestion and the sequencing reactions as
specified below.
Restriction digestion
1. Transfer 5 µl of your plasmid DNA into a clean microcentrifuge tube marked E
for EcoR I digestion. Transfer another 5 µl of your plasmid DNA into a
second microcentrifuge tube marked U for the uncut control. Add your initials
and section letter onto the side or top of the tube for later identification.
2. Add 15 µl of EcoR I restriction digestion mix to the tube labeled ‘E’. Add 15 µl
of Mock restriction digestion mix (buffers but no enzyme) to the tube labeled
‘U’. This mock digestion serves as a negative control to determine if the
EcoR I digestion worked.
41
Sequencing reaction
1. Transfer 10 µl of your plasmid DNA into a clean 0.5 ml (small) microcentrifuge
tube marked with your initials and section letter on the top.
2. Add 2 µl of primer to the tube.
3. Add 8 µl of BigDye TerminatorTM reagent to the tube. This reagent is a
commercially-obtained mix containing a modified Taq DNA polymerase,
buffers, MgCl2, dNTPs and labeled ddNTPs. Close the cap and place the
tube in the ice bucket on your bench.
Your reaction tubes will be collected by your instructor and will be placed in
the thermal cycling machine along with those from students in your section for
the sequence cycling reaction. When the cycling is complete, they reaction
products will be precipitated and sent to the sequencing facility in Rebstock
for the automated reading of your sequence. The sequencing results will be
sent to your lab instructor and you will have the opportunity to analyze them in
the ‘Genomics and DNA Sequence Homology Analysis’ lab.
42
Learning to Clone DNA
Part Three: Restriction Analysis
Background:
Bacterial restriction enzymes were originally discovered as part of a
defense system against infection by bacterial viruses. These restriction enzymes
function to break up or hydrolyze pieces of DNA with specific DNA recognition
sequences. This activity is useful in preventing infection because the bacteria
can partially hydrolyze the DNA of an infecting virus, if the virus has the
sequence that is recognized by the restriction enzyme. You can see, then, that
having one restriction enzyme does not completely prevent a bacterium from
infection by bacterial viruses because each restriction enzyme only cleaves the
DNA of viral strains that contain the specific DNA recognition sequence. To
increase their resistance to infection, many bacteria synthesize multiple
restriction enzymes.
Restriction enzymes are named for the organism from which they are first
isolated and characterized. The first three letters of the enzyme name comes
from the name of the bacterial species, and is optionally followed by a Roman
numeral indicating the order in which this enzyme was isolated from that
organism relative to other restriction enzymes from the same organism. The
name part is usually pronounced rather than spelled out, but the Roman numeral
is always pronounced by the cardinal number (one, two, three, etc.). For
example, EcoRI (pronounced: E-coh-are-won) is derived from E. coli strain R,
and it was the first enzyme isolated from this strain; Bgl II (bagel-too) was the
second restriction enzyme isolated from Bacillus globigii.
In today’s lab we will separate our restriction digest by gel electrophoresis.
We will continue with lecture and when our samples have run far enough to
analyze the results we will talk about them as a class.
Experimental Protocol:
1. After you’ve retrieved your restriction digest from last class, spin the
microcentrifuge tube briefly to bring any condensed liquid down to the
bottom. Add 3 µl of loading dye.
2. Load the entire sample onto the gel.
3. Run all the samples in a 1.5% agarose gel for ~1hour at 120 volts.
43
Genomics and Homology Analysis
Background:
Often times when doing biological research, an investigator clones a gene,
sequences it, and using the internet, discovers that it has never been
characterized before. While it is exciting to discover a new gene further research
becomes complicated by the fact that the function of this new gene product is not
likely known. This problem can be approached in a number of different ways, the
simplest of which is to look at gene sequences from other organisms and
compare them with the sequence of the novel gene. If a gene with a similar
sequence is found, and if the function of that similar gene product has already
been described, it might give a clue as to the function of the newly discovered
gene product. In this way, a scientist might be able to hypothesize about the
function of the new gene based upon its homology with another species’ gene(s)
of known function. In today’s lab, you will learn to analyze DNA sequences
relative to other known sequences. We will be looking at the sequence data we
obtained from the cloning experiment from the past few weeks.
Procedure Part A:
Searching for information about your cloned DNA
What gene is it? Is it a gene at all?
1. Go to the Saccharomyces Genome Database at www.yeastgenome.org
2. Select the BLAST program from the links along the bar at the top of the
page. When the BLAST page opens, you should see a box to enter your
sequence data. You can just copy and paste the sequence results (in
FASTA format) into that box. Next, change the Filter to “none.” Finally, hit
“Run BLAST” to scan your sequence against the yeast genome.
FASTA format and the BLAST program will be explained in class. When
you submit your sequence for BLAST, the program will search the DNA
sequence of the entire yeast genome to find the sequence you obtained
from cloning. When it gets a match, a page will open with the results of
your match and will contain links that you can use to find out more about
the sequence that your clone matches (for example, gene name, location
in the genome, possible function etc.)
3. On the results page, you will see some matching scores as well as a
probability number (P-value). You can think of the P-value as the
probability that the hit you found was found by random chance and is not
significant (so you want the P-value to be very low!). Start filling out the
Sequence Information Form as you complete the following steps.
44
4. It is hard to see much information about the sequence on this page, so
select “ORF Map” to see a map of the open reading frames in this area of
the genome.
5. The match to your sequence is located between the red dotted lines. If
there is a red or blue box in that location, you have cloned part of a gene.
The key to the colors used in this diagram is directly below the diagram.
The gene names are short combinations of letters and numbers near the
red or blue box. If the dotted lines do not intersect with a gene, skip to
step 7. We will talk more about non-coding DNA in the next activity.
6. Click on the colored box representing the gene, and a new page will come
up with information about that gene. Use this information to answer the
questions about the gene product on the Sequence Information Form.
7. If you have not cloned part of a gene, open a sequence file from List A (a
folder on the course webpage) and repeat steps 1 – 6 on your own, filling
out the Sequence Information Form as you go. If your clone WAS part of
a gene, open a sequence file from the “List B” folder. Repeat steps 1 – 6
with this sequence on your own and fill out the Sequence Information
Form for this sequence.
Procedure Part B:
Why didn’t everyone clone a gene?
Which organism has more non-gene DNA, yeast or
humans?
1. We will use “Map Viewer” from the NCBI website to explore the yeast and
human genomes. Go to http://www.ncbi.nlm.nih.gov/mapview/
2. Once at this site, select “saccharomyces cerevisiae” from the phylogenetic
tree. You should now be viewing an interactive map of the yeast
chromosomes.
3. Select one of the sequences you studied from the Sequence Information
Form. Click on the chromosome number where this sequence was
located. From this screen record the number of base pairs shown as well
as the total number of genes.
4. As a class, we will perform a calculation to determine the percent of base
pairs in the chromosome that are part of a gene. Next, we will repeat
steps 2 – 4, looking at a human chromosome. Are humans or yeast more
efficient with their use of DNA?
45
Human Genome vs. Yeast Genome
Fact Table:
Number of
chromosomes
Total size (bp)
Total genes
(estimated)
Repetitive elements
Pseudogenes
% of genome that is
coding
Human
22 + X and Y
Yeast
16
3,000,000,000
~25,000
12,067,000
~6,200
43%
~8,000
~1%
10%
48 (non-gene ORF’s)
~50%
The average length of a gene is ~1000 bp (although some yeast genes are much
shorter and some human genes are much longer). Based on this length, what is
the maximum number of genes the human and yeast genomes could have?
Which genome is closer to having its maximum number of genes?
46
Sequence Information Form
Sequence Cloned in Class
Sequence information:
Name of file
P-value of top hit from the BLAST
search
Chromosome
and coordinates (bp)
Gene name
Crick or Watson strand?
Gene product:
What is the function of the gene
product?
Where is the gene product found in
the cell?
Can yeast survive without this
gene?
47
Chymotrypsin Enzyme Activity Assay
By April Bednarski
Introduction:
Chymotrypsin is a digestive enzyme that can break peptide bonds in
proteins. It is part of a class of enzymes called proteases. Other members of
this class are involved in blood clotting, development, and inflammation.
Proteases are present in plants as well as animals. Papain is another protease
and can be isolated from papayas.
In our bodies, the substrate for chymotrypsin is a specific type of peptide
found in proteins that contains a bulky hydrophobic group. In this experiment,
the activity of chymotrypsin will be measured using the substrate, GPNA
(glutaryl-L-phenylaniline 4-nitroaniline). When chymotrypsin cleaves the peptide
bond in GPNA, p-nitroaniline (PNA) is released. PNA is a yellow substance, so
we can use UV spectroscopy to detect product formation. At the end of each of
your assays, you should notice that the solution becomes more yellow.
Enzymatic reaction:
O
OH
NO2
O O
HN
H
N
H
NO2
Chymotrypsin
+
GP
H2 N
GPNA
(Glutaryl-L-phenylalanine 4-nitroaniline
p-nitroaniline
(absorbs strongly at 410 nm
General Procedure:
Each enzymatic assay will be set up with a total volume of 5 mL in a
cuvette (or test tube) and activity will be measured by monitoring the absorbance
of each assay at 410 nm in a Spectronic 20 spectrophotometer. Each assay will
contain a final concentration of 10 µM chymotrypsin in 100 mM sodium
phosphate buffer (NaH2PO4). For each assay, first combine the substrate and
buffer in the cuvette and mix well. Set the cuvette in the Spec 20 and set the
A410nm to zero. Initiate the reaction and start timing as you add the enzyme (0.5
mL of chymotrypsin). Record the A410nm at 1, 2, 3, 4, and 5 minutes.
For each assay, the substrate concentration will increase. We will see
how the amount of substrate added affects enzyme activity. Do you think you will
see an infinite increase in enzyme activity as you add more and more substrate?
Why might the enzyme activity reach a constant maximum level?
48
Procedure:
Mix the NaH2PO4, DMF, and substrate for each assay in a cuvette according to
the table below. DO NOT ADD ENZYME YET! Lab each cuvette with the assay
number (A1 – A5). Use buffer which has been pre-warmed to 30˚C.
(each assay will total 5 mL):
Assay #
NaH2PO4 (pH
(substrate
7)
concentration)
A5 (1.5 mM)
4 mL
A4 (1.2 mM)
4 mL
A3 (0.9 mM)
4 mL
A2 (0.6 mM)
4 mL
A1 (0.3 mM)
4 mL
DMF
Substrate
in DMF
Enzyme
0.0 mL
0.1 mL
0.2 mL
0.3 mL
0.4 mL
0.5 mL
0.4 mL
0.3 mL
0.2 mL
0.1 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
0.5 mL
Set up the enzyme, assay cuvettes, and data table next to the spectrophotomer.
Make sure the spectrophotometer is warmed up and has been zeroed. For each
assay, follow the procedure below: (Note: after adding the enzyme, work as
quickly as you can since the reaction has started!)
1. Place cuvette in the Spec 20, set to zero.
2. Add the enzyme, gently mix by shaking the cuvette lightly, place back in
the Spec 20, and start timing.
3. Record A410nm at 0 min, 1 min, 2 min, 3 min, 4 min, and 5 min in the data
table below.
4. After 5 min, you can stop timing. Repeat this procedure with the next
assay.
Data Table:
Assay #
A1
A2
A3
A4
A5
Substrate
conc.
0.3 mM
0.6 mM
0.9 mM
1.2 mM
1.5 mM
A410nm
0 min
A410nm
1 min
A410nm
2 min
A410nm
3 min
A410nm
4 min
A410nm
5 min
From this table, plot A410nm (y-axis) versus time (x-axis) for each assay. You will
have a total of five lines on your plot (each assay is a different line). Label each
line. Use this plot to answer the questions on your lab write-up. Hand in this plot
with your write-up.
49
Data Plot
50
51
Biology 280 Lab Write-up: Chymotrypsin Assays
Name:________________________
Date of Investigation:
1. Which assay reached maximum absorbance the quickest and which assay
took the longest?
2. What do you predict the line on the plot would look like for an assay with 3
mM substrate? Describe below, or lightly sketch on your plot. Explain
your answer.
3. Why would differences in substrate concentration lead to a difference in
enzyme activity? Hint: think about what needs to happen for the enzyme
to make product out of the substrate.
4. The slope of each line on your plot starting at (0,0) and extending along
the assay line for as long as it is straight, is called initial velocity and is
represented by V0. What trend can you see from comparing the slope of
this line for each assay?
52
Yeast Genetics
Adapted from Modern Genetics for All Students, Washington University in St. Louis, Science
Outreach, 2000. T137, S112
Background:
Today we will do a real genetic experiment, with a real, live model organism. The
model organism we will use is baker’s yeast. Yes, this is the same organism that
the bakers of Bunny Bread use to make that spongy white stuff for your peanut
butter and jelly sandwiches! Baker’s yeast is a unicellular fungus with a life cycle
that at first seems very different from that of the more familiar animals and plants.
As different as it looks at first glance however, you will notice that the yeast life
cycle does resemble the life cycle of animals and plants in several very important
regards. It involves haploid and diploid cells and thus meiosis and recombination.
Indeed, yeast follows the same basic rules of inheritance that we do, even
though its haploid cells bear no resemblance to the sperm and egg cells of
animals.
Because yeast can complete its life cycle in less than a day (under optimum
conditions), it can be used to perform many different genetic experiments in a
very short time. As a result, it is one of the most popular model organisms for
geneticists who are interested in studying the basic principles of genetics that
apply to all eukaryotic organisms.
In this exercise, we will use red and white strains of yeast to study a simple
genotype-phenotype relationship that until now we had only encountered as a
theoretical concept. In addition to strains that differ in color, we will also need
strains that differ in mating type so that they will be able to fuse to make diploids.
Yeast geneticists call the two mating types of yeast a (small a) and _(alpha). But
to be sure that we do not confuse the two strains in our experiment, we will use
the terms A (capital A) and _(alpha). In a sense, the two mating types, A and _,
are to yeast as males and females are to animals.
Your job will be to formulate a hypothesis about the genetic basis for the color
difference that you will observe between the haploid strains you will cross, use
that hypothesis to make a prediction about what color the diploids will be, and
then mate four strains of yeast to test your hypothesis.
THE LIFE CYCLE OF YEAST
Haploid and diploid yeast cells look similar, and both can divide mitotically to
form large colonies. Haploid cells come in two mating types, which we will call A
and _. As long as these two mating types are kept apart, they continue to grow
and divide in the haploid state. But if they make contact, they fuse to form an A/_
diploid. Under most conditions, the A/_ diploids will grow and divide continuously,
forming colonies.
Under certain nutritional conditions, however, A/_ diploids undergo
meiosis to produce new A and _ haploids. If the A and _ cells are not separated
at once, they fuse again to make new A/_ diploids. At first this might seem like a
53
waste of effort, but it is not.
Because various alleles will have been sorted out and recombined at random in
the process, the new generation of A/_ diploids will include individuals that are
genetically different from those in the earlier generation, and one or more of
these new variants might be better adapted to the nutritional conditions that now
exist.
Procedure:
Mating Type A
R
Mating Type A
W
R
R
R
W
W
W
1
2
3
4
Making the Genetic Crosses
Do not open your petri dish until instructed to do so.
1. With the dish still closed, draw four circles and number them 1 to 4 (fig. 3).
2. Get a sterile toothpick. Lift the agar-containing part of the petri dish, and —
while keeping the dish upside down — gently rub the Red Mating type A colony
with the end of the tooth pick. Now rub the toothpick on the agar lightly in the
center of circle 1. You want to deposit only a small number of cells on the agar —
barely enough to see. If you have more than this, try to scrape off the excess.
Discard the toothpick in the waste jar.
3. With a new sterile toothpick gently rub the Red Mating type _ colony and then
rub the toothpick in the same region of circle 1 where you rubbed with the Red
Mating type A toothpick. Mix the two kinds of yeast cells together with the
toothpick, but avoid stabbing the agar. Discard the toothpick in the waste jar.
4. Repeat steps 3 and 4 for the three empty circles, so as to cross the White
Mating type A and Red Mating type _ strains in circle 2, the Red Mating type A
and White Mating type _ strains in circle 3, and the two white strains in circle 4.
5. Place your petri dish in the incubator or where your teacher directs you.
54
Biology 280 Lab Write-up: Yeast Genetics
Name:________________________
Date of Investigation:
Part 1:
1. Describe the appearances of the four colonies of haploid yeast cells at the
beginning of the experiment.
2. Formulate a hypothesis about the genetic difference that causes the difference
in appearance of the red and white yeast strains.
3. Which color trait do you think will be dominant, or do you think that they will be
codominant? Why?
4. Based on the above hypotheses, what do you predict the color phenotypes of
the diploids will be in the following four crosses that you have set up?
Red Mating type A x Red Mating type
Red Mating type A x White Mating type
White Mating type A x Red Mating type
White Mating type A x White Mating type
55
Part 2
Observing Your F1 (A/_ Diploids)
1. Get your petri dish and observe your results. Even though there are no sperm
and eggs involved, a Punnett square diagram can be used to record and analyze
the results of yeast crosses such as the ones you performed. Use your observed
results to fill in the blanks on the Punnett square below.
Mating Type α
Mating Type A
R
W
R
W
Genotype______________ Genotype______________
Phenotype_____________ Phenotype ____________
Genotype______________ Genotype______________
Phenotype ____________ Phenotype ____________
2. Are any of your results unclear? If so, indicate which ones, describe what you
see, and provide a good explanation for these results.
3. What ratio of phenotypes did you observe as a result of the four crosses you
performed?
4. What does this indicate about which allele is dominant and which is recessive?
5. Is this what you predicted?
56
6. In the table below, list what you predicted and what you observed for each of
the four crosses.
Cross
Predicted Phenotype of
Diploid
Observed Phenotype of
Diploid
Red Mating type A by
Red Mating type α
Red Mating type A by
White Mating type α
White Mating type A by
Red Mating type α
White Mating type A by
White Mating type α
6. If your predicted and observed phenotypes do not agree, how can you account
for that, and can you propose a good hypothesis to account for the results you
actually observed?
7. If you have come up with a new hypothesis, can you think of a way to test it?
57
58
Reebops: Inheritance of Traits
Adapted from Modern Genetics for All Students, Washington University in St. Louis, Science
Outreach, 2000. T119, S90
Introduction:
Reebops are imaginary creatures that were invented by Patti Soderberg at the
University of Wisconsin. We think that this is a good “model” system for studying
heredity. As you create baby Reebops from marshmallows and other objects,
they can help you see how the visible traits of a baby are related to the
combination of genes that it inherited from its mom and dad (and why all the kids
in the family don’t always look alike) Have fun Reebopping!
Procedure:
1. You and your lab partner will receive an envelope that contains 14 red
chromosomes that belong to Mom Reebop and 14 green chromosomes that
belong to Dad Reebop. Decide which of you will act as Mom and which will act
as Dad. Place your chromosomes on the table in front of you, letter side down.
Your lab partner should do the same with the other set of chromosomes.
2. Arrange your 14 chromosomes into pairs by length and width. Select one
chromosome from each of your seven pairs and place all seven in a special
“gamete” (egg or sperm) pile. Your lab partner should do the same. The leftover
chromosomes should now be returned to the envelope.
What type of cell division has just occurred?
3. Combine the seven red and seven green chromosomes from the two gamete
piles to form a “baby” pile. Now each Reebop baby will have 14 chromosomes
just like Mom and Dad did. But half will be red and half green, indicating that half
came from Mom and half from Dad.
4. Line up the chromosomes contributed to the baby by Mom and Dad in pairs of
similar size, letter side up. You will see that each chromosome in a pair carries a
gene of similar type (same letter of the alphabet).
Some chromosome pairs might carry the same allele (either both capital letters
or both lower case), indicating that the baby is homozygous (has two alleles of
the same type) for the kind of gene carried on that chromosome.
Other chromosome pairs might carry one dominant (capital letter) allele and one
recessive (lower-case) allele, indicating that the baby is heterozygous (has two
alleles of different type) for the kind of gene carried on that chromosome.
59
The combination of genes carried on these seven chromosome pairs defines
your Reebop baby’s genotype (genetic constitution). Record this genotype on the
lines below.
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
5. Refer to the Reebop Genotype-Phenotype Conversion Table on page S94 to
determine your baby’s phenotype. Record the phenotype on the lines below,
keeping the phenotypic traits in the same order as the genes you listed in step 4.
____________
____________
____________
____________
____________
____________
____________
6. You are now ready to construct your Baby Reebop. Collect the body parts that
you will need and return to your bench to build your baby.
Genotype Phenotype
DD
Dd
dd
Three body segments
Three body segments
Two body segments
AA
Aa
aa
Two antennae
One antenna
No antennae
NN
Nn
nn
Red nose
Orange nose
Yellow nose
EE
Ee
ee
Two eyes
Two eyes
One eye
MM
Mm
mm
Three green humps
Two green humps
One green hump
TT
Tt
Curly tail
Curly tail
60
tt
Straight tail
LL
Ll
ll
Blue legs
Blue legs
Red legs
Note: Toothpicks function as the bones and ligaments that hold the Reebops
together.
Reebop Problem Solving:
1. Define the following terms and give an example of each from this activity.
(You may refer to the Genetic Glossary.)
allele:
genotype:
phenotype:
homozygous:
heterozygous:
2. If a Reebop female with a red nose and a Reebop male with a yellow nose
marry and have children, what genotype and phenotype for nose color will their
children have? (You may refer back to the Reebop Genotype-Phenotype
Conversion Table.)
genotype ______________________________
phenotype______________________________
3.
If a Reebop female with one antenna and a Reebop male with no
antennae marry and have children, what genotypes and phenotypes might their
children have with respect to number of antennae?
genotype ______________________________
phenotype______________________________
4.
If a Reebop female with one antenna and a Reebop male with one
antenna marry and have children, what is the probability that they will have a
baby with no antennae?
5.
If a Reebop female with two green humps and a Reebop male with two
green humps marry and have children, what is the probability that their first baby
will have two green humps?
61
6.
If a Reebop female with three green humps and a Reebop male with three
green humps marry and have children, what is the probability that they will have
a baby with two green humps?
7.
If a Reebop baby has a straight tail, but both of his parents have curly
tails, what are genotypes of the two parents?
Class Reebop Data Collection:
Fill in the NUMBER of Reebops found in your class with the following heritable
traits:
Antennae
Nose color Humps
One
_____ Red
_____ One
Two
_____ Orange
_____
_____ Two
_____
None _____ Yellow_____ Three _____
Eyes
Segments
Tail
Leg color
One
_____ Two
_____ Curly _____ Blue _____
Two
_____ Three _____ Straight_____
Red
_____
62
Biology 280 Lab Write-up: Analysis of Reebop Findings
Name:________________________
Date of Investigation:
1. Describe the phenotypes of Mom and Dad Reebop.
2. Using the information in the Reebop Genotype-Phenotype Conversion Table,
list all the possible genotypes that would produce the phenotypes exhibited by
Mom and Dad.
3. How many of the Reebop babies in your class have the same phenotypes as
Mom or Dad?
4. Do any two babies in your class have exactly the same phenotypes?
5. Why do some Reebop babies have traits that are not seen in either Mom or
Dad?
6. Which Reebop traits are dominant?
7. Which Reebop traits exhibit codominance?
8. Use the information you have about the phenotypes of all of the Reebop
babies in your class to figure out what the genotypes of Mom and Dad Reebop
are.
9. If you know the genotype of the parents, is it possible to predict all of the
possible genotypes of babies that they might produce?
63
10. If you know the genotype of the parents, is it possible to predict the genotype
of any particular baby, such as their first one?
11. The Reebops appear to have only one gene on each chromosome. Do you
think this is true of real, living organisms?
64
Can Sunscreen Stop Mutations?
Adapted from Modern Genetics for All Students, Washington University in St. Louis, Science
Outreach, 2000. T184, SP164 and WU Bio 2960 Spring 2004 Lab Manual.
BRING IN YOUR OWN SUNSCREEN FOR THIS LAB, IF YOU LIKE.
UV as a Mutagen
You will use ultraviolet light (UV) to induce mutations in bacteria. UV is
present in sunlight as radiation that (as the name suggests) is just beyond the
violet end of the visible spectrum. UVis absorbed by, and damages, DNA, and
therefore it is a powerful mutagen (mutation-causing agent) and carcinogen
(cancer-causing agent). Fortunately, however, the ozone layer of earth's upper
atmosphere filters out most of the UV that streams toward earth from the sun. If
the ozone layer were to disappear, the surface of the earth would become
uninhabitable. Indeed, some scientists believe that the drastic decline in frog and
salamander populations that has been observed around the world in the past ten
years is probably a result of the thinning of the ozone layer. It is also believed
that this thinning has been caused by chlorofluorocarbons (CFCs), such as
Freon, which were once used as refrigerants and propellants in hair-spray cans.
Even at present ozone levels, the ability of organisms to live at the surface of the
earth is due to their remarkable ability to repair DNA damage that has been
caused by UV. The importance of DNA repair is highlighted by a genetic disease
called xeroderma pigmentosum (XP) in which a critical enzyme required for DNA
repair is nonfunctional. People with XPcharacteristically have many skin
abnormalities, and most of them die of skin cancer before age 20.
As important as they are, however, our DNA repair systems are
inadequate to fully protect us against the carcinogenic effects of the UV rays in
sunlight or tanning salons. It is now recognized that a single sunburn as a child
or teenager can increase the risk of skin cancer many years later.
Because UV is absorbed by the top few layers of skin cells in humans,
however, UV radiation cannot penetrate to our ovaries or testes, where our germ
cells are stored. Thus, UV damage in humans is largely restricted to cells in the
skin and eyes. Although it is the principal cause of human skin cancer, UV does
not cause heritable mutations that can be passed on to our offspring. In contrast,
small one-cell organisms, such as bacteria, do not have the benefit of a
protective layer of skin, and any non-fatal UV-induced mutations that they
experience are invariably passed on to their progeny. We will take advantage of
that fact in this exercise to study the mutagenic effects of UV on bacteria.
How do the mutations occur?
Nucleic acids absorb UV light strongly, and UV irradiation of DNA results
in a wide variety of chemical alterations. One of the most important of these
alterations is the formation of covalent bonds between adjacent pyrimidine
residues on a strand of DNA, resulting in the formation of pyrimidine dimers.
65
As shown in Figure 1 below, the pyrimidines in such dimers are connected by
cyclobutane rings. Pyrimidine dimers present a severe problem to the cell,
because they block the progression of DNA replication and transcription. As you
might expect, mechanisms have evolved to remove pyrimidine dimers from DNA.
These repair mechanisms include general excision repair and a second
mechanism, called photoreactivation. In the latter, pyrimidine dimers are split
apart by an enzyme that utilizes energy from visible light. This photoreactivating
enzyme (PRE; DNA photolyase) is one of very few proteins known in biology
able to absorb and utilize the energy in visible light.
Figure 1: A thymine dimer
As in the tobacco experiment, you will be working with the Gram-negative
bacterium Serratia marcescens. You will monitor the same two effects of the
mutagenesis, death and loss of the red pigment, prodigiosin.
Procedure:
1. Label 8 NA-plates as follows: 0 Dark, 15 Dark, 30 Dark, 0 Light, 15 Light,
30 Light, 30 Sunscreen. The numbers refer to the number of seconds of
UV short-wave light exposure. For the 30 sunscreen plate only, draw a line
through the center of the back of the plate; label one side +SS and the other SS (to designate with/without sunscreen). Write your initials on each plate.
2. Spread 100 µl of the suspension of Serratia bacteria onto the surface of each
of your labeled plates.
3. Arrange your plates by exposure time and take them to the UV lamp for
irradiation. For the "30 Sunscreen" plate, place a piece of sunscreen-coated
Saran wrap over the plate so that the sunscreen coating covers the +SS half
of the plate. Irradiate the sunscreen plate along with the "30 dark" and “30
light" plates. Note: remove the plastic lids from the plates before
irradiation.
4. After UV exposure, the "dark" plates will be placed directly in a dark, room
temperature incubator; the "light" plates will be incubated at room
temperature in the presence of white light for ≥ 6 hours. After this initial light
exposure all plates will be incubated in the dark at room temperature.
66
5. After the plates incubate at room temperature for about 48 hours, they will be
placed in a refrigerator to arrest colony growth. Next week you will examine
the plates and complete the analysis of this experiment.
67
68
Biology 280 Lab Write-up: Effectiveness of Sunscreen
Name:________________________
Date of Investigation:
1. What was the purpose of exposing the bacterial cells to UV light?
2. What do the numbers 10, 12 and 15 refer to in our mutagenesis experiment?
3. A) What is the most common kind of mutation caused by UV light exposure?
B) What repair mechanisms are available to repair this kind of mutation?
4. What was the purpose of putting some plates in the dark and some plates in
the light?
69
5. Describe the control that was used in the UV mutagenesis experiment.
Complete Questions 6 –8 after you obtain your results.
6. Do you think the Saran wrap had an effect on the experiment? How could
you find out?
7. Describe your results. Include number of colonies and frequency of white
colonies.
Control plate:
0 Dark
0 Light
10 Dark
10 Light
15 Dark
15 Light
15 + Sunscreen
15 - Sunscreen
8. Discuss your results. Describe whether the results were expected. Offer
possible explanations for any unexpected results. Finally, assess the sunscreen
for the ability to protect against UV- caused mutations.
70
Learning to use Bioinformatics tools.
This lab is designed around bioinformatics tools that are widely used in
biomedical research today. Over the past few years, the number of freely
available software programs and web-based research tools has increased
dramatically. Knowing how to use these tools is very important to research and
health professionals in order to access and interpret the constantly growing
amounts of genomic and proteomic information. These bioinformatics tools as
well as genomic information are freely available on the web to anyone who
knows how to access them. Bio280 introduces these computer-based research
tools in a context that reinforces concepts presented during the Bio280 lecture.
You will be given your own gene to study during the 3 lab sessions. This
gene will be the focus of all your guide sheets and final report. There will be one
other person working on the same gene in each lab section. You are
encouraged to help each other, but work individually. All the projects are similar
in their structure and format, even though they are designed around different
genes. The basic process for using web-based research tools is the same for all
projects.
71
List of Projects:
Lung Cancer
K-Ras
Cytochrome P450 1A1
Apoptosis
Caspase 1
Superoxide Dismutase 1
Metabolic Disease
Phenylalanine Hydroxylase
Hypoxanthine-guanine phosphoribosyltransferase
Cholesterol Biosynthesis
HMG-CoA Reductase
Low Density Lipoprotein Receptor
Mitochondrial Diseases
ATP Synthase 6
Cytochrome c Oxidase 1
Report:
You will be compiling printouts from each lab period into a report showing
what you have found out about your gene using the web-based resources. You
will also write a summary of your findings. This report will be due at the last lab
session
Report format:
Final report should contain:
1. Title page with:
Your name and date
Protein name
Lab section number
2. Summary (1 page, single-spaced, see “Sample Summary”
on the 3055 Website)
3. Multiple sequence alignment (annotated)
4. Swiss-Pdb Viewer figures with labels
5. Don’t include anything extra
72
Laboratory 1 - Introduction
COX-2 (PTGS2) Tutorial
On the first day of lab, we will be working through a tutorial on the
web-based bioinformatics programs that you will be using for your
research projects. You will also be receiving your project packet, which
will contain the introduction, specific directions, and reading assignments
for your project. There are ten possible projects, and you will be working
on a project that is the same as one of the people at the computer next to
you. Although you must each complete your own research project, you are
encouraged to collaborate, discuss your projects, and help each other
during the lab.
The tutorial is based on the enzyme cyclooxygenase-2 (COX-2), which
also has the name prostaglandin synthase-2 (PTGS2). You can read more
about this protein on the next page. In this tutorial, the bioinformatics tools from
the NCBI (National Center for Biotechnology Information) website will be
introduced. NCBI is a division of the National Institute of Health (NIH). These
tools include Gene, GenBank, RefSeq, and PubMed. Gene is a database of
genes in which each entry contains a brief summary, the common gene symbol,
information about the gene function, and links to websites, articles, and
sequence information for that gene. GenBank is a historical database of gene
sequences, which means it contains every sequence that was published, even if
the same sequence was published more than once. Therefore, GenBank is
considered a redundant database. RefSeq is a database of sequences that is
edited by NCBI and is NON-redundant, meaning that it contains what NCBI
determines is the strongest sequence data for each gene.
Finally, we will be learning to use ClustalW, which is a multiple sequence
alignment program. It allows you to enter a series of gene or protein sequences
that you believe are similar and may be evolutionarily related. These sequences
are usually obtained by performing a BLAST search. ClustalW then aligns the
sequences, so that the lowest number of gaps is introduced and the highest
numbers of similar residues are aligned with each other. ClustalW uses a
scoring matrix similar to BLOSUM-62, which is explained in your text and will be
presented in lecture.
73
Introduction to COX-2 (PTGS2)
The enzyme we will be focusing on has two names. It is called
prostaglandin H2 synthase-2 and cyclooxygenase-2 (COX-2). COX-2 has been
thoroughly studied because of its role in prostaglandin synthesis. Prostaglandins
have a wide range of roles in our body from aiding in digestion to propagating
pain and inflammation. Aspirin is a general inhibitor of prostaglandin synthesis
and therefore, helps reduce pain. However, aspirin also inhibits the synthesis of
prostaglandins that aid in digestion. Therefore, aspirin is a poor choice for pain
and inflammation management for those with ulcers or other digestion problems.
Recent advances in targeting specific prostaglandin-synthesizing enzymes have
lead to the development of Celebrex, which is marketed as an arthritis therapy.
Celebrex is a potent and specific inhibitor of COX-2. Celebrex is considered
specific because it doesn’t inhibit COX-1, which is involved in synthesizing
prostaglandins that aid in digestion. This is a remarkable accomplishment given
the great similarity between COX-1 and COX-2. This achievement has paved
the way for developing new therapies that bind more specifically to their target
and therefore have fewer side effects.
Understanding the enzyme structures of COX-1 and COX-2 helped
researchers develop a drug that would only bind and inhibit COX-2. Many of the
types of information and tools used by researchers for these types of studies are
freely available on the web. In this tutorial, and throughout this lab course, you
will be introduced to the databases and freely available software programs that
are commonly used by professionals in research and medicine to study genes,
proteins, protein structure and function, and genetic disease.
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GenBank Entry
Date of latest
modification
The unique name for the gene
locus. It was originally designed
to help group entries with
similar sequences. The first
letter usually is related to the
organism.
Number of
base pairs
Molecule type
Organismal
division of
GenBank
A unique number for
the sequence.
A new GI number is
given if the sequence
changes in any way.
Publication that
discusses the data in
the entry.
Information about the
gene sequence.
CDS = nucleotide
coding sequence
Base count (A,T,G, and C) and actual sequence follow.
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SwissProt Entry
SwissProt Accession Number
Click here before printing
76
SwissProt Entry Continued:
Guide sheet questions focus on “Comments” and “Features”
Sections:
Amino acid residue numbers
Active site residues,
disulfide linkages,
residues bound to
carbohydrates, etc.
Mutations
found
Sources
disagree
Best sequence
known for this
protein
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The Sequence Manipulation Suite:
Clear contents of the box
Submit sequence
Choose reading frame for translating
Paste FASTA formatted
nucleotide sequence from
Word document here.
Obtain FASTA formatted translation of nucleotide sequence
78
Taken from http://www.ebi.ac.uk/clustalw/index.html
ClustalW Submission Form:
We want the human sequence on top,
so output should be in order of input,
not order of aligned. Change this to
“input.”
Choose “full”
Align with numbers, so we
can find particular residues in
the alignment.
No color for easier
printing.
Enter FASTA formatted
sequences from Word document
using “copy” and “paste.”
Choose “Run” to submit.
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Background on PTGS1 (Cox-1) and PTGS2 (Cox-2):
Follow these directions to access the entries for PTGS1 and
PTGS2 in the “Gene” database at the NCBI Website:
A. First, go to the NCBI homepage using the link on
the lab webpage, or by going to:
http://www.ncbi.nlm.nih.gov
B. Select “Gene” from the database pulldown menu.
Type “PTGS” in the search box, then click “Go.”
C. Scan the results for the “Homo sapiens” entries.
There should be one called “PTGS1” and one
called “PTGS2.”
D. Select each entry by clicking on its name, then
read the paragraph under the “Summary” section
for each entry.
Read the “Summary” section for both of these genes, then
answer the questions below.
1. PTGS1 and PTGS2 are isozymes. Isozymes
catalyze the same reaction, but are separate genes.
What types of reactions do PTGS enzymes
catalyze? Also, what pathway are these enzymes a
part of?
2. How is the expression of PTGS1 and PTGS2
different?
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Part 1 – Getting sequence information and viewing database entries
NCBI – Gene
1. Go back to the “Gene” entry for Homo sapiens PTGS2.
2. What is the gene name?
3. What is the GeneID number?
4. Where in the human genome is this gene located?
5. What is the RefSeq accession number for the mRNA sequence of
Homo sapiens prostaglandin-endoperoxide synthase
2?__________________.
6. What is the RefSeq accession number for the Homo sapiens PTGS2
protein sequence (called “product”)?_________________.
7. Open the RefSeq protein entry, then choose “FASTA” from the pulldown menu. Copy the sequence (including the title line designated
by the “>” symbol) and paste it into a Word document.
8. In Word, select the “Replace” tool under the EDIT menu. In the
“find” box, type “^p” to find all paragraph marks. Don’t type
anything into the “replace” box. Then click “Replace All.” This will
eliminate all the paragraph marks in the document. If you still see
white spaces in the sequence, use the same procedure, but type
“^w” in the “find” box to represent white spaces.
9. You now should add back a paragraph mark (hit return) after the title
line (that starts with “>”) and before the sequence starts. Save the
file as PTGS2prot.doc on your desktop.
Note: Please review the entry for “FASTA” in the Glossary (at the end of
this manual). Understanding this definition will be very important for
working with the bioinformatics programs. To learn more, go to:
http://www.ncbi.nlm.nih.gov/blast/fasta.shtml
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Swiss-Prot Entry
10. Go to the Expasy website and search for the Swiss-Prot entry for PTGS2.
(Hint: use the gene name to search and be sure to select the HUMAN
protein from the search results).
11. Write at least three alternate names for this protein.
12. Where in the cell is this protein located?
13. What types of drugs target this protein?
Sequence Manipulation
14. Go to the Sequence Manipulation Suite
(http://bioinformatics.org/sms/).
15. Click on “Translate” under “DNA Analysis” heading from the menu.
16. Clear the data entry box by hitting “Clear”.
17. Copy the mRNA sequence from your Word file and Paste it into the
data entry box on the Sequence Manipulation website.
18. Select “Reading Frame 3” and “direct” from the pull-down menus,
then click “Submit”.
19. When the Output window opens, review it and make sure it doesn’t
contain too many stop codons (which are represented with a “*”).
20. Compare this sequence with the sequence in the “PTGS2prot.doc”.
What are the first residues that are the same in the sequences? Do
the sequences look like they are the same? (Hint: protein
sequences should start with a methionine, M.)
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Part 2 – Multiple Sequence Alignment with ClustalW
21. On the course website under “COX2 Tutorial”, there should be a file called
“ClustalWseq”. Click on this link to download the pdf file to your desktop.
To open the file in Acrobat Reader, hold the curser over the file on your
desktop, hold down the “Control” key on your keyboard as you click and
hold with the mouse. A list of programs should appear on your desktop.
Highlight “Adobe Acrobat,” then release the mouse. The file should open
in Adobe Acrobat. If you have the file open in “Preview,” you will not be
able to select multiple pages of text at the same time, so close the file and
open it in Acrobat Reader as described above. Using the “Text select”
tool, select all the text and copy or go under the “Edit” menu and “Select
All.” This file contains six FASTA formatted sequences of PTGS2 from
different organisms. The top sequence is the human PTGS2 protein
sequence you have been working with.
22. Go to the ClustalW website and enter (by using “copy” and “paste”) all of
the FASTA formatted sequences into the data entry box. Select “input” for
the Output order so the human sequences will stay at the top in the
alignment. Press “Run.”
23. Copy the alignment and paste it into a Word document. To make this file
readable, do the following things:
a. Go to “Page Set-up” under “File” and change the page orientation
to landscape.
b. Select all text and change to “Courier” font, size 10. Courier is the
best font for alignments because all the letters are the same width.
c. Save this file to the desktop as “ClustalW.doc”
24. Review the alignment. What symbols are used for positions in the
alignment that contain identical, highly homologous, homologous, and
non-homologous residues? Are the residue numbers mentioned in steps
14 and 15 conserved? Why would you expect them to be conserved?
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Laboratory 2 - Project Research
Guide Sheet 1
Working with Primary Protein Structure Information
The research projects begin by using some of the same tools you used
last week in the COX-2 tutorial. Guide Sheet 1 will provide directions for the
web-based research you will be doing today. First you will search for your gene
in the Gene database. Gene will contain the RefSeq sequence for your protein,
which you will download in FASTA format. FASTA format is defined in your
Glossary. Be sure to review this definition before you begin on Guide Sheet 1.
You will continue to learn about your protein using the SwissProt database. The
SwissProt database is a database maintained by the Swiss Bioinformatics
Institute and contains entries for thousands of proteins. You can search for the
protein you are studying by using the gene name given in Gene. The SwissProt
entry contains some of the same information that you found in Gene, but also
contains a lot of information about the protein sequence, structure, and function
that is summarized in a fairly easy-to-read format.
The ultimate goal for today’s lab is to create a multiple sequence
alignment for your protein using ClustalW. You will use this alignment to identify
the protein mutation, to observe regions of high sequence conservation, and to
map secondary structure predictions. The protein mutation is important to
identify since it is the basis for your project and is important for understanding the
link between the protein and the disease you are studying. The regions of high
sequence conservation are important because they often correspond to regions
in the protein that are important to the protein’s function. Next week, you will be
studying the crystal structure of your protein and will be able to check the
accuracy of the secondary structure predictions that you mapped onto your
alignment. The program used for making the secondary structure predictions is
called PSIPRED. If your protein is a membrane protein, you will also have
MEMSTAT predictions, which predict which regions of your protein, are
imbedded in a membrane.
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Guide Sheet 1
Part 1 – Obtaining the basics: Getting sequence information and viewing
the SwissProt and GenBank entries for your protein
Directions: Follow this guide sheet and answer the questions in your
project packet that accompany this guide as you work through each
section. Be sure to refer to hints specific to your project in your project
packet.
Translating your patient’s cDNA
1. Obtain the mutant cDNA sequence from the course website. Open
the file and copy the sequence.
2. Go to the Sequence Manipulation Site
(http://bioinformatics.org/sms/).
3. In the menu to the left, Click on “Translate” found under the heading
“DNA analysis”.
4. Clear the search box, then paste your patient’s cDNA sequence into
the search box. Choose a reading frame from the pull-down menu
(the project manuals indicate which reading frame to choose). Click
“Submit.”
5. You should be able to find the sequence of your protein by finding
the first methionine (M), then continuing until you see the first “*”
which is a stop codon. Copy the protein sequence in that region,
starting with the first “M” and paste it into a word document. Save
the document in your folder on the desktop. Now you have saved
the file of the mutant protein sequence.
NCBI – Gene
6. Using Gene on the NCBI website, find the entry for the protein you
are studying by searching with the protein name. Be sure to select
the Homo sapiens protein from the list of results. Answer question
1.
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7. Open the entry for the RefSeq protein sequence and save the sequence in
FASTA format to your desktop. Name this file “wildtypeprot.doc” so that
you will know it is the un-mutated protein sequence.
Swiss-Prot Entry
8. Go to the ExPASy website and search for the SwissProt entry for your
protein using either the protein name the gene name. Be sure to select
the human protein from the list of results. Make sure the information in the
entry is the same as you saw in the Gene entry. If your protein is an
enzyme, the EC number is a good way to double-check. You may want to
record the SwissProt entry number in case you want to find this entry
again.
Part 2: BLAST sequence: Finding homologous proteins
Protein-protein BLAST
9. Perform a BLAST search using the RefSeq protein sequence (the unmutated protein sequence). First, select PSI-PHI BLAST. Then paste the
FASTA formatted protein sequence in the search box. Select the nrprotein database. Click “BLAST” to begin. On the next page that
appears, select “Format.” You may need to wait a few minutes before the
results page opens. After obtaining the results, choose 5 sequences from
various positions in the results. The goal is to choose a variety of
sequences that differ in evolutionary distance from the human protein. Be
sure not to choose any sequences that are human, since they are the
same as your search sequence. For each of the five sequences, click on
the sequence name to view the GenBank entry for the sequence. Then
view the sequence in FASTA format. Copy and paste all the FASTA
formatted sequences into the same Word file and save it to the desktop.
At the beginning of this file, add your mutant protein sequence, also in
FASTA format.
10. This Word file will be used to create the multiple sequence alignment, so
the formatting is very important. The format for this file should be like the
example used in the tutorial for COX-2. Review the entry for FASTA
format in the Glossary. You should end up with a Word file that contains
the 5 sequences from the BLAST search plus the un-mutated human
protein sequence and your mutant sequence for a total of 7 sequences.
Each sequence should be in FASTA format and contain a title line
(starting with >, then text, then a return). Shorten the text to contain JUST
the species information so it will fit in the alignment (next step). For
example, you should erase the “gi” line and add in something simpler like
“pig,” “cow,” etc. Your mutant sequence should read “>mutant”. At the
86
end of each title, be sure to press return to separate it from the rest of the
sequence.
Part 3 – Multiple Sequence Alignment
11. Go to the ClustalW website and enter (by using “copy” and “paste”) all
your FASTA formatted sequence into the data entry box. The default
parameters will work for us, except for the output order.
a. Select “input” for the Output order
b. Press “run”
12. Save the alignment by copying and pasting the alignment into a Word
document. At first the alignment will look broken up. Follow these steps
to make it readable again.
a. Select all
b. Change the font to size 10 and Courier
c. Change the page set-up to landscape
d. Save the file to your desktop
13. Scroll through the alignment and make sure none of the blocks of
sequences are separated by a page break. Save and print the alignment.
We will be working with this alignment next week and it will be part of your
final report.
14. On your alignment, compare the wild-type protein sequence with your
mutant sequence and mark any differences that you observe.
Secondary Structure Predictions – using PSIPRED
15. To save time, the PSIPRED and MEMSTAT predictions for your protein
are already available for you in the same format as we received them.
Obtain these results from the course website under your project name and
print them out. The Glossary provides more information on how to submit
requests for PSIPRED and MEMSTAT predictions if you want to use these
tools outside this course.
From this lab, you should save the multiple sequence alignment for your
Final Report. The multiple sequence alignment should have the secondary
structure predictions and the mutation mapped.
NEXT WEEK: Do the structure problem set for next week. It’s important to
view the problem set online from the lab website in order to see the
structures in color!
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Laboratory 3 - Project Research
Guide Sheet 2
Investigation of the Crystal Structure
The goal of this lab is to analyze the crystal structure for your protein in
order find the functionally important areas of your protein and predict the effect of
the mutation on protein function. You may determine that the mutation makes
the protein more active, less active, or that the mutation is likely to have no effect
of protein function.
To do these investigations, we will be using the protein structure-viewing
program called DeepView/Swiss-Pdb Viewer. This program has some
similarities to programs you have used in previous course, like Chime, and
Protein Explorer. DeepView can easily model mutations and is easy to learn to
use in one day. It is not a web-based program, but both the Mac and PC
versions can be downloaded for free from the ExPASy website. There are also
DeepView tutorials available for free from the ExPASy website. At the beginning
of lab, a brief tutorial on how to use DeepView will be given using the COX-2
protein.
The first step today is to obtain the data file of the crystal structure for your
protein. In some cases, the crystal structure has not been solved for your exact
protein, so you will analyze the structure and model the mutation in a
homologous protein. In this case, the amino acid numbering will be slightly
different, but details are given in your project packets. The data files for crystal
structures are called pdb files and are all stored in the Protein Data Bank. You
can search the Protein Data Bank website for your protein and download the pdb
file to your desktop.
In analyzing the crystal structure, the focus is on identifying the
noncovalent interactions (Van der Waals, H-bonds, and ionic bonds) of the amino
acid side chain before and after it is mutated. How do the interactions change or
how are they maintained when the amino acid is mutated? Be sure to review
both the distances and nature of these non-covalent interactions before coming
to this lab.
Crystal structures are the results of experiments, and so it is important to
consider experimental error when analyzing a crystal structure. One way
experimental error is reported in a crystal structure is by giving the resolution.
Resolution of a crystal structure is the accuracy of the prediction of each atom
location. For example, if the resolution is 2 angstroms, you can be confident that
the atom is located within a 2 angstrom radius of where it is shown in the pdb file.
This is important to consider when measuring distances, since each distance
measured in a crystal structure is actually ± the resolution of the crystal structure.
88
Tool Bar
Transverse
Movement
(along plane
of screen)
Rotate
Zoom
Distance Tool measure the
distance between
two atoms in
Angstroms
Identity Tool - the
residue type and
number will appear
in the display for the
atom you select
Radius Tool - the side
chains of residues within a
certain distance (you
specify) will appear
around the atom you
select.
Re-center Tool - the
display will re-center
around the atom you select
and will rotate around that
atom when you use the
rotate tool.
Mutate Tool - When you
click on this tool, then an
atom in the display, you can
mutate a side chain to
another type and it will
appear in the display in the
lowest energy conformation
89
Panel under : “Prefs”
“Ribbons”
Make sure there is a check in
this box to show a cartoon
ribbon in the display
In this panel, you can choose a different color for each side of the 3-dimensional ribbon as well as
different colors for the helices, sheets, and coils (loops). There should be checkmarks as shown here
for each color box. When you click on the “Color” button, a panel appears to choose a color. It is a
good idea to choose the same color for each of the top, bottom and sides of all helices. You don’t
need to change any of the other settings in this panel for this course. Click “OK” when finished.
90
Control Panel
This column
needs to be
checked to
show a
substrate or an
amino acid
side chain.
Pdb file name
Display side
chain of
amino acid
Show label for
residue and
number in display
Show
spacefilling
dots for this
residue
Chain
secondary structure:
h = helix
b = beta sheet
Checkmarks
make residues
show up in
ribbon format
in the display
amino acid three
letter code
residue number
Clicking on the box and
selecting a color will
color the amino acid.
Leaving it as is will be
cpk color mode. (see
Glossary)
91
Guide Sheet 2
Obtaining 3D Structure Information:
Searching for Structure Files (pdb files):
Data files that contain the three-dimensional coordinates for protein structures
are called “pdb” files. Pdb files are named in 4 characters (numbers and letters).
These files are stored in the Protein Data Bank (www.rcsb.org/pdb). You can
perform a search to find the structure you are looking for either from the NCBI
website using their “Structure” database, or search directly at the Protein Data
Bank website. The search results are a little easier to understand from the NCBI
website. If you know exactly what you are looking for, you can directly search
the Protein Data Bank.
Follow the directions in your project manuals to get directions for finding the pdb
file and analyzing the structure in Swiss-Pdb Viewer/DeepView.
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Laboratory 4 - Project Research
Guide Sheet 3
Investigating the biological impact of the mutation - using the
OMIM and KEGG databases
For this lab session, your research will focus on investigating the biological
impact of the mutation you are studying. To do this, you will use the OMIM and
KEGG websites. OMIM stands for the Online Mendelian Inheritance in Man
database. The OMIM database was started at John Hopkins University and is
now maintained by NCBI and can be found through a link on the NCBI
homepage. The OMIM database contains entries for both diseases with known
genetic links and entries for the genes that have been linked to a disease. Each
OMIM entry is a summary of the research that has been performed on the
disease or gene and contains links to the research articles that it summarizes.
You will be able to read about the clinical and biochemical research that has
been performed related to the mutation you are studying. Each link in the OMIM
entry will open an abstract from the PubMed database. PubMed is a literature
database, and is also maintained by NCBI. PubMed is a searchable database of
medical and life science journal articles. Most of the abstracts for these articles
can be accessed through PubMed, but in order to access the entire article, you
need to go to each individual journal website and have a subscription to the
journal. The WashU library system has subscriptions to electronic versions of
many of these journals that you can access through the E-journal link on the
WashU library home page. Most journals have their articles available online as
.pdf files for articles published between 1995 to present. However, the older
articles must still be accessed through the paper versions stored in libraries.
The other database that you will be using is the KEGG database. KEGG
stands for the Kyoto Encyclopedia of Genes and Genomes. It is a database of
metabolic pathways that is maintained by a research institute in Japan. It
contains all the known metabolic and signaling pathways. Each protein in the
pathway and each small molecule metabolite (ex. ATP) has its own entry in the
database that can be accessed by clicking on the protein or metabolite in the
pathway figure. By using this website, you can make predictions about what
would happen to downstream events in the pathway if the protein you are
studying is either less active or more active.
In order to write the summary for your final report, you will need to use the
information from this lab’s research as well as what you learned from studying
the crystal structure of your protein in order to draw a conclusion about what the
biological impact is of the mutation you are studying. This conclusion should be
supported by the web-based research you have performed, but will most likely
require further clinical and biochemical research in order to be proven or
disproved.
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Guide Sheet 3
Using Web-based resources to investigate the biological impact of
the mutation and its possible role in human disease
OMIM search: The OMIM (Online Mendelian Inheritance
of Man) database contains short, referenced reviews
about genetic loci and genetic diseases. It can be a very
useful resource for finding out what type of research
has been done on a gene or a disease.
KEGG search: The Gene entry for your gene that you viewed during the first
day of your research (Guide Sheet 1) will contain a link for the KEGG pathway(s)
related to your protein. Scroll down to the “Additional Links” and select the
“KEGG pathway” link. KEGG stands for Kyoto Encyclopedia of Genes and
Genomes. The www.kegg.com site contains a database of metabolic maps.
Follow the directions in your project manuals to answer Guide Sheet 3 questions.
94
Glossary.
BLAST – Basic Local Alignment Search Tool – A program that compares a
sequence (input) to all the sequences in a database (that you choose). This
program aligns the most similar segments between sequences. BLAST aligns
sequences using a scoring matrix similar to BLOSUM (see entry). This scoring
method gives penalties for gaps and gives the highest score for identical
residues. Substitutions are scored based on how conservative the changes are.
The output shows a list of sequences, with the highest scoring sequence at the
top. The scoring output is given as an E-value. The lower the E-value, the
higher scoring the sequence is. E-values in the range of 1^-100 to 1^-50 are
very similar (or even identical) sequences. Sequences with E-values 1^-10 and
higher need to be examined based on other methods to determine homology. An
E-value of 1^-10 for a sequence can be interpreted as, “a 1 in 1^10 chance that
the sequence was pulled from the database by chance alone (has no homology
to the query sequence).”
This program can be accessed from the NCBI homepage or:
http://www.ncbi.nlm.nih.gov/BLAST
Reference: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J.
(1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410.
BLOSUM – Block Scoring Matrix - A type of substitution matrix that is used by
programs like BLAST to give sequences a score based on similarity to another
sequence. The scoring matrix gives a score to conservative substitutions of
amino acids. A conservative substitution is a substitution of an amino acid
similar in size and chemical properties to the amino acid in the query sequence.
Discussed in the Berg text, p.175 – 178.
Bioinformatics - Bioinformatics is a field of study that merges math, biology, and
computer science. Researchers in this field have developed a wide range of
tools to help biomedical researchers work with genomic, biochemical, and
medical information. Some types of bioinformatics tools include data base
storage and search programs as well as software programs for analyzing
genomic and proteomic data.
ClustalW – A program for making multiple sequence alignments.
http://www.ebi.ac.uk/clustalw/index.html
W. R. Pearson (1990) “Rapid and Sensitive Sequence Comparison with FASTP
and FASTA” Methods in Enzymology 183:63 - 98.
95
Conserved – when talking about a position in a multiple sequence alignment,
“conserved” means the amino acid residues at that position are identical
throughout the alignment.
Conservative residue change – when talking about a position in a mulitple
sequence alignment, a “conservative change” is when there is a change to a
homologous amino acid residue.
cpk coloring mode - This coloring mode colors based on atom identity:
red = oxygen
blue = nitrogen
orange = phosphorous
yellow = sulfur
gray = carbon
DeepView/Swiss-Pdb Viewer – a program for viewing 3-D structures. It loads
“.pdb” files, which contain the 3-D coordinates for molecular structures. SwissPdb Viewer is easy and free to download on any computer (Mac or PC) and can
be used no matter what Browser you are using. It is fairly easy to learn to use at
the basic level, however, it also has very advanced capabilities that can be useful
in research. It is also a nice program to use with PovRay, which allows you to
make graphic files from pdb information. This is important when making figures
for a presentation, report, or journal article. If you would like to download SwissPdb Viewer for your own computer, the program is available for free and is easy
to download from the website, “us.expasy.org/spdbv”. A help manual is also
available here if you have further questions that aren’t addressed in this course.
http://us.expasy.org/
To run this program with Mac OSX, you must first change the monitor settings.
a. Open “System Preferences” on your computer.
b. Double click on the “Displays” icon.
c. On the right-hand side of the panel, choose “thousands” of colors
from the list (changing it to “thousands” from “millions”).
d. Then close System Preferences and then open Swiss-Pdb Viewer.
Names of some other structure viewing programs:
RasMol (www.openrasmol.org)
Kinemage (www.kinemage.biochem.duke.edu)
Protein Explorer (www.proteinexplorer.org)
EC number - Enzyme Committee number - Given by the IUBMB (International
Union of Biochemistry and Molecular Biology) classifies enzymes according to
the reaction catalyzed. An EC Number is composed of four numbers divided by
96
a dot. For example the alcohol dehydrogenase has the EC Number 1.1.1.1
ExPASy – Expert Protein Analysis System - A server maintained by the Swiss
Institute of Bioinformatics. Home of SWISS-PROT, the most extensive and
annotated protein database. The Swiss-Pdb Viewer protein-viewing program is
also available at this site for free download.
http://us.expasy.org/
FASTA – A way of formatting a nuleic acid or protein sequence. It is important
because many bioinformatics programs require that the sequence be in FASTA
format. The FASTA format has a title line for each sequence that begins
with a “>” followed by any needed text to name the sequence. The end of
the title line is signified by a paragraph mark (hit the return key).
Bioinformatics programs will know that the title line isn’t part of the sequence if
you have it formatted correctly. The sequence itself does NOT have any returns,
spaces, or formatting of any kind. The sequence is given in one-letter code. An
example of a protein in correct FASTA format is shown below:
>K-Ras protein Homo sapiens
MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDI
LDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVP
MVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRK
HKEKMSKDGKKKKKKSKTKCVIM
To learn more, go to:
http://www.ncbi.nlm.nih.gov/blast/fasta.shtml
GenBank - a database of nucleotide sequences from >130,000 organisms. This
is the main database for nucleotide sequences. It is a historical database,
meaning it is redundant. When new or updated information is entered into
GenBank, it is given a new entry, but the older sequence information is also kept
in the database. GenBank belongs to an international collaboration of sequence
databases, which also includes EMBL (European Molecular Biology Laboratory)
and DDBJ (DNA Data Bank of Japan). In contrast, the RefSeq database (see
entry) is non-redundant and contains only the most current sequence information
for genetic loci. The GenBank database can be searched at the NCBI
homepage:
http://www.ncbi.nlm.nih.gov/
Gene – an NCBI database of genetic loci. This database used to be called
LocusLink. Entries provide links to RefSeqs, articles in PubMed, and other
descriptive information about genetic loci. The database also provides
information on official nomenclature, aliases, sequence accession numbers,
phenotypes, EC numbers, OMIM numbers, UniGene clusters, map information,
97
and relevant web sites. Access through the NCBI homepage by selecting “Gene”
from the Search pulldown menu.
Genome – The entire amount of genetic information for an organism. The
human genome is the set of 46 chromosomes.
Homologous – When referring to amino acids, a homologous amino acid is
similar to the reference amino acid in chemical properties and size. For example,
glutamate can be considered homologous to aspartate because both residues
are roughly similar in size and both residues contain a carboxylic acid moiety
which gives them similar chemical properties.
KEGG – Kyoto Encyclopedia of Genes and Genomes – This website is used for
accessing metabolic pathways. At this website, you can search a process, gene,
protein, or metabolite and obtain diagrams of all the metabolic pathways
associated with your query. You will see a link to the KEGG entry at the end of
the Gene entry for a gene.
http://www.genome.ad.jp/kegg/
NCBI – National Center for Biotechnology Information – This center was formed
in 1988 as a division of the NLM (National Library of Medicine) at the NIH
(National Institute of Health). As part of the NIH, NCBI is funded by the US
government. The main goal of the center is to provide resources for biomedical
researchers as well as the general public. The center is continually developing
new materials and updating databases. The entire human genome is freely
available on this website and is updated daily as new and better data becomes
available. The NCBI homepage:
http://www.ncbi.nlm.nih.gov
NCBI also maintains an extensive education site, which offers online tutorials of
its databases and programs:
http://www.ncbi.nlm.nih.gov/About/outreach/courses.html
OMIM - Online Mendelian Inheritance in Man – a continuously updated catalog of
human genes and genetic disorders, with links to associated literature
references, sequence records, maps, and related databases. Access through
the NCBI homepage or:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM
Protein Data Bank – (PDB) – A database that contains every published 3-D
structure of biological macromolecules. It contains mostly proteins, but also DNA
and RNA structures. Also see RCSB.
http://www.rcsb.org/pdb/
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A pdb file is a file containing the three-dimensional coordinates (x,y,z) for each of
the atoms in the protein. This type of file is made using the data obtained from
either an X-ray crystallography experiment or an NMR experiment. Once you
have pdb file of a protein, you can open the file in various structure viewing
programs to view the protein structure.
Proteome – the entire set of expressed proteins for an organism. This term is
commonly used to discuss the set of proteins that are expressed in a certain cell
type or tissue under specific conditions.
PSIPRED – a server for predicting secondary structure from protein sequences.
The predictions are made based on a database of known secondary structures
for protein sequences. These predictions are estimated to be correct ~80% of
the time. This server can also be used to predict transmembrane segments.
http://bioinf.cs.ucl.ac.uk/psipred/
McGuffin LJ, Bryson K, Jones DT. (2000) The PSIPRED protein structure
prediction server. Bioinformatics. 16, 404-405.
Jones DT. (1999) Protein secondary structure prediction based on positionspecific scoring matrices. J. Mol. Biol. 292: 195-202.
PubMed – a retrieval system containing citations, abstracts, and indexing terms
for journal articles in the biomedical sciences. PubMed contains the complete
contents of the MEDLINE and PREMEDLINE databases. It also contains some
articles and journals considered out of scope for MEDLINE, based on either
content or on a period of time when the journal was not indexed, and therefore is
a superset of MEDLINE.
http://www.ncbi.nlm.nih.gov/
RCSB – Research Collaborative for Structural Bioinformatics – A non-profit
consortium that works to provide free public resources and publication to assist
others and further the fields of bioinformatics and biology dedicated to study of 3D biological macromolecules. Members include Rutgers, San Diego
Supercomputer Center, University of Wisconsin, and CARB-NIST (at NIH).
RefSeq - NCBI database of Reference Sequences. Curated, non-redundant set
including genomic DNA contigs, mRNAs, proteins, and entire chromosomes.
Accession numbers have the format of two letters, an underscore bar, and six
digits. Example: NT_123456. Code: NT, NC, NG = genomic; NM = mRNA;
NP = protein (See NCBI site map for more of the two letter codes).
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Sequence Manipulation Suite – a website that contains a collection of webbased programs for analyzing and formatting DNA and protein sequences.
http://bioinformatics.org/sms/
SNP = Single Nucleotide Polymorphism.
synonymous change– The nucleotide change results in NO change in
amino acid.
non-synonymous change – The nucleotide change DOES result in a
change in amino acid.
heterozygosity – A measure of the genetic variation in a population with
respect to one locus. Stated as the frequency of heterozygotes for that
locus.
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Website List for Bio 280
http://www.nytimes.com
The Tuesday edition of the New York Times has a Science section. You can
also search for past science and health-related articles at this site. You have to
register, but registration is free.
http://www.madsci.org/libs/libs.html
A resource for getting started using science resources on the WWW.
http://science.education.nih.gov/
NIH educational resources on many genetics and health-related topics.
http://www.genome.gov
Homepage for the National Human Genome Research Institute (NHGRI)
http://www.doegenomes.org/
Genome program of the U. S. Department of Energy Office of Science: There is
a lot of helpful information about different genome projects.
http://www.the-scientist.com/home
This website offers coverage of the latest developments in life sciences research,
technology and business. You have to register, but registration is free.
http://www.sciam.com/
This is the web of Scientific American magazine which contains good reviews of
current research in science and health and is written for a broad audience.
http://www.sciencemag.org/
This is the Science magazine which publishes the most up-to-date discoveries of
scientific fields. Written for a scientific audience.
http://www.nature.com/
This website is the Nature magazine. Both the Science and the Nature cover the
newest scientific information. Written for a scientific audience.
http://www.cell.com/
The Cell magazine contains a lot of latest discoveries of biological fields. Written
for a scientific audience.
http://www.science.gov/
Use the browsable index for a broad range of topics from Agriculture and Food to
Science Education. Searchable by keyword.
http://science.howstuffworks.com/
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Searchable database for simple explanations on a wide range of topics, including
cell biology, DNA science, and genetics. Easy to understand. A good place to
start, but explanations are aimed at a K – 12 audience.
http://www.biology.arizona.edu/
Online resources to supplement biology courses.
http://www.cellsalive.com/toc.htm
Animations for cell biology.
http://www.accessexcellence.org/AB/GG/
A searchable database of curriculum tools for K – 16.
http://www.hhmi.org/biointeractive/
Howard Hughes Medical Institute online curriculum resources on health-related
topics.
http://www.hgc.gov.uk/business_consultations2maintext.rtf
Background information for identification of individuals: Unit 1
A discussion document on the storage, protection and use of personal genetic
information from Great Britain.
http://www.biology.washington.edu/fingerprint/dnaintro.html
This page was created as a class project at the University of Washington to
provide to the Internet basic information on the structure and function of DNA as
it relates to DNA fingerprinting.
http://whyfiles.org/
Searchable database for articles on the “science behind news.”
http://www.sciencenews.org/
A weekly online news magazine from the journal Science. News on current
research written for a broad audience.
http://www.geneticalliance.org/DIS/
A public information search tool for genetic disease information
http://www.bbc.co.uk/go/science/news1a/-/genes/
Track the major developments in gene science since Charles Darwin published
"The Origin of Species":
http://www.nabt.org/sub/contact/reps.asp
The National Association of Biology Teachers (NABT)
http://www.nsta.org/chapters
The National Science Teachers Association (NSTA)
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http://www.exploratorium.edu/edunews
From the Exploratorium in San Francisco. Aimed at K-12 students and teachers.
http://sciwebserver.science.mcmaster.ca/biology/CBCN/genetics/mac_dnafp.htm
A short article about DNA fingerprinting and conservation genetics
http://www.blackwell-synergy.com/links/doi/10.1046/j.15231739.1994.08030744.x/abs/
A short abstract which demonstrates uses for DNA fingerprinting in conservation
genetics
http://www.rothamsted.bbsrc.ac.uk/notebook/courses/guide/
A Beginner's Guide to Molecular Biology. Great pictures and explanations for
basic molecular biology concepts.
http://jura.wi.mit.edu/bio/education/BOA-2000/left.htm
Has several great links to background information in DNA science and protein
science.
IMPORTANT:
Note that all of the above web sites are maintained by academic
sources, government agencies, or reviewed journals. These sources
generally subject the work that they post to peer review, and are
usually reliable- although mistakes do occur! Be aware that many
other web sites are set up by people who do not set a high standard
for accuracy, or who have a particular opinion on a topic. Please
check the source of any web site that you use as a reference for your
papers.
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GENETIC GLOSSARY
allele: one of two or more forms of a gene that can exist at a single locus.
chromosome: a structure in the nucleus of a eukaryotic cell that contains a
linear array of many genes. A chromosome is composed of a single DNA double
helix molecule wound around many protein molecules that stabilize it and
regulate its function.
codominant: refers to a pair of alleles, both of which exert an effect on the
phenotype when they are present together. In codominance, the heterozygote
has a phenotype different from that of either homozygote and sometimes (but not
always) is intermediate in phenotype.
diploid: having two complete sets of chromosomes, one set derived from the
mother and one from the father.
dominant: refers to an allele that has the same effect on the phenotype
whether it is present in the homozygous or heterozygous condition. (Thus, if A is
a dominant allele, individuals with the AA and Aa genotypes have the same
phenotype.)
genotype: the specific combination of alleles that an individual possesses at
one or more loci.
haploid: having only one set of chromosomes (as in a sperm or egg nucleus).
heterozygous: having two different alleles at a particular locus.
homozygous: having two identical alleles at a particular locus.
incomplete dominance: a form of codominance in which the heterozygote
is about half-way between the two homozygotes in phenotype. (For example, if
homozygous plants have red or white flowers and the heterozygous plant has
pink flowers, the situation is sometimes called incomplete dominance. But it is
just a special type of codominance.)
locus: a region of a chromosome or DNA molecule where a particular kind of
gene, coding for a particular kind of protein, is located. Different variants at a
single locus are known as alleles.
meiosis: the “reduction division” in which a diploid cell divides to produce
haploid cells that will function as gametes (eggs or sperm).
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phenotype: the outward appearance of an individual with respect to one or
more traits that is associated with some particular genotype.
recessive: refers to an allele that has no effect on the phenotype unless it is
present in the homozygous condition.
recombination: the process in which two haploid sets of chromosomes are
brought together in a pair of gametes to produce a new diploid offspring. Usually
this new diploid will be different in genotype from both of its parents.
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