AP Biology Lab-03

Lab 3
Comparing DNA Sequences to Understand

Evolutionary Relationships with BLAST
Big Idea 1: Evolution
How can bioinformatics be used as a tool to determine evolutionary

relationships and to better understand genetic diseases?
Please be sure you have read the

student intro packet before you do this lab.
(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)
Lab Investigations Summary

Pre-lab and Questions: Cladograms
Part A - Structural Cladogram

Part B - Biochemical Analysis for Cladogram Construction
GAPDH gene and protein percentage similarities
Part C - Biochemical Analysis of Cytochrome c
Cytochrome c protein enzyme percentage of difference in amino acid
sequences between 17 organisms
Lab Investigation 3.1
Part 1 - BLAST Practice

BLAST practice with an unknown fossil specimen
Cladogram Prediction and Tutorial using BLAST
Part 2 - Student Guided Inquiry
Student guided inquiry using BLAST to query a gene responsible for
producing a mutant disease-causing protein of interest
Tutorial on using Entrez Gene to obtain nucleotide sequence for gene of
interest
Copyright 2013 Quality Science Labs, LLC
LAB 3 - Comparing DNA Sequences to

Understand Evolutionary Relationships
with BLAST
Big Idea 1 Evolution
How can bioinformatics be used as a tool to determine evolutionary relationships
and to better understand genetic diseases?
BACKGROUND
How can we apply science to the study of origins? In the past, this meant
the application of historical science and interpretations of the complex and variable
fossil records and dating methods. What evidence do we have to support observable
science when it comes to the evolutionary changes in populations and gradual, linear,
primitive-to-advanced taxonomic groups of organisms? Are we relying on conjuring
up explanations for observations or are we using the scientific methodology to form
testable hypotheses to build on the evolutionary theory? The following is a quote
from Washington State University, School of Biological Sciences (July 2008; Mack,
R.N. and Black, R. A.)
Although essential, the formation and testing of hypotheses (sometimes
called strong inference) can be a formidable exercise. Observations must be
reduced to simple hypotheses: a Null Hypothesis that says that a factor has
no effect in causing an observed phenomenon and a companion Alternate
Hypothesis that states the factor is having an effect. Equally important
is the recognition that multiple hypotheses must be formulated and tested
together. The overall goal is to disprove or falsify each of the multiple
hypotheses. This approach seems initially an odd choice, but it arises because
few hypotheses can be proven. Among an initial group of hypotheses, the
hypothesis that cannot be disproved is taken as the likely explanation. Use
of multiple hypotheses avoids unconsciously becoming the guardian of a pet
hypothesis the unfortunate consequence of having only one hypothesis
is that it may become either a so-call Expandable Hypothesis (in which
the original hypothesis is simply expanded to account for all new evidence,
often as exceptions or special cases) or worse, a Ruling Theory (in which
facts are deliberately sought out that support the theory while conflicting
facts are attacked or ignored).
Consider how (or if) a multitude of testable hypotheses can be applied

to using bioinformatics to determine evolutionary relationships and genetic
diseases. Molecular genetics has opened up a quantitative world of sequencing
genes (DNA) and gene products (proteins). The thirteen-year Human Genome
Project (HGP) mapped over 20,000 genes from a human. With sequencing of
genes and proteins in many different organisms, we now have evidence showing
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changes in the genetic material of different taxonomic groups of organisms and

their populations. Molecular Evolutionary Geneticists create models to analyze
and reveal the evolutionary relationships among organisms and populations. As
gene sequences are identified that relate to specific genetic diseases in a particular
organism, similar sequences can be quickly compared in the powerful BLAST data
base. If all but one hypothesis explaining a phenomenon has been disproved or
falsified, the remaining hypothesis is considered the best current approximation
of the truth. The operative word here is current because any hypothesis (even if
elevated to the rank of a theory, i.e., a hypothesis for which some evidence has been
assembles) is subject to continual review and testing. (July 2008; Mack, R.N. and
Black, R. A.)
What do we know about human DNA and gene sequencing? Thirteen years
of sequencing human DNA (Human Genome Project HGP) has mapped the
nucleotide sequences of over 20,000 gene sites in humans. This information has
been made public to scientists all over the world.
How can knowing the human genome help in understanding genetic
diseases? If gene sites for genetic diseases can be compared to nucleotide sequences
of the same normal human gene site (from HGP data), then science can progress
to alleviating these diseases. Gene therapy is the use of DNA to treat disease.
Scientists introduce genes into human cells via a bacterial or viral vector (a bacteria
or a virus that integrates a foreign gene into their DNA and transfers it to the host
DNA), focusing on diseases caused by single-gene defects, such as cystic fibrosis,
hemophilia, muscular dystrophy, thalassemia, and sickle-cell anemia. Today, most
gene therapy studies are aimed at cancer and hereditary diseases linked to a genetic
defect.
How can molecular information help in developing evolutionary
relationships? Biological evolution is looking to biochemistry to provide evidence
of gradual change between taxonomic groups. Comparing sequencing of DNA
nucleotides or amino acids in proteins, they hope to create trees of life showing
ancestral relationships. Cladograms are branching diagrams used to illustrate
what is predicted to be the successive points of species divergence from common
ancestral lines. Traditional cladograms
were generated largely on the basis of
physical characteristics but have been
partially discarded for more recent
ones developed using DNA and RNA
sequencing information, which is now
very commonly used in the generation of
cladograms. The Pre-labs in this unit will
give you practice in developing your own
cladograms from anatomical information
(physical characteristics).
Consider
if cladograms based on molecular
sequencing provide irrefutable evidence
Lemurs killed in Madagascar for bushmeat
to prove evolutionary development
and the tree of life? This is for you to think about as you view the biochemistry
databases of comparing cytochrome c and decipher the basic assumptions inherent
in the cladogram diagram in the Pre-labs. Then Lab Investigation 3.1 will use
molecular information to develop the cladogram.
Are similarities in biochemical molecules evidence of evolution? For

biological evolution, similarities are a major foundational evidence for evolution.
According to Darwinian theory, it stands to reason that the larger the number
of similarities between two organisms, the closer their evolutionary relationship
is likely to be. Physical characteristics are not always easy to discern, have proven
unreliable, and the fossil record has not provided conclusive evidence. However,
today we have access to DNA sequencing, gene sites, and protein amino acid
sequencing in data bases easily accessible to the world. You will examine the
molecular evidences in this lab and ask the following: Has the molecular evidence
proved gradual evolutionary progression from one taxonomic group to another and
is there evidence for the intermediate or transitional species?
In accordance with our promise to be open-minded and consider other
interpretations, another perspective based on similarities could be considered.
What if the reason for similarities in structure, function, and a biochemical
base was that it allowed all living organisms to fit into a common ecological web
and a food chain for survival?
Molecular sequencing, implications, and assumptions: Protein sequencing
looks at sequences of amino acids. Cytochrome c oxidase (an enzyme involved
in mitochondrial electron transport for ATP production) is a protein with gene
locus (COX1) that has been studied in depth. The amino acid sequencing is
known for many organisms. In fact, it is used today in DNA barcoding to identify
unrecognizable carcasses as to whether they are endangered species in an attempt
to control the Bushmeat crisis in third world countries.
As mentioned previously, the cytochrome c enzyme has been studied
extensively and amino acid sequencing is known in several organisms. This
facilitates comparisons on a molecular level among several different phyla. In a
comparison of 17 organisms ranging from humans to photosynthesizing bacteria,
cytochrome c has been analyzed and compared. Is there evidence of progressively
more divergence as biological evolution would predict? Logically, Darwinian
evolution would expect progressively more divergence on the molecular level as we
move up the evolutionary scale from silkworms to humans. When comparing living
organisms, it would be reasonable to predict a greater molecular distance from the
insect to the amphibian than to the living fish, greater distance still to the reptile,
and greater than that to the mammal. This pattern is not found. If fish evolved
into amphibians in the evolutionary scenario, one would expect the cytochrome c
amino acid sequencing in fish to be more similar to the cytochrome c sequencing in
amphibians. But these are not the cases. What is found in the variety of vertebrates
is the same or similar % difference in sequencing percent sequence distance of
each cytochrome c protein. Instead of amphibians being closer to fish (having
a smaller % difference in sequencing) as would be predicted in the evolutionary
scale, on a molecular level amphibians are no closer to fish than to reptiles or
mammals. Hence, this is still a puzzle to solve.
Has evidence of intermediate species been found in molecular sequencing?
If intermediate species are considered (bridges transitioning between amphibians
and reptiles), Darwinian evolution would predict some species of amphibians to
be closer to fish (more primitive species) and others to be closer to reptiles (more
advanced species). This is also NOT the case in cytochrome c analysis. All
vertebrates from horses, rabbits, chickens, and turtles to bullfrogs have the
same percent distance of differences in cytochrome c sequencing from that in
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fish (carp). Another puzzle to be solved.

What is the perspective and what are the assumptions underlying cladogram
construction? At this time, biological evolution has high expectations that
biochemistry will provide evidence of gradual change between taxonomic groups.
They are counting on gene site DNA sequencing and protein amino acid sequencing
to provide information for a progressive sequence on which to base progressive
evolutionary changes from one taxonomic group to another; from bacteria to
invertebrates to vertebrates.
BLAST: Evolutionary connections is a data base used to detect similarities and
differences in genomes. BLAST stands for Basic Local Alignment Search Tool. Using
BLAST, you can input a gene sequence of interest and search entire genomic libraries
for identical or similar sequences in a matter of seconds. Students will use BLAST to
compare several genes and then using the information, construct a cladogram, such as
commonly used, to show the implied evolutionary relatedness of an unknown fossil.
Students will also use Entrez Gene website and BLAST in Part 2 of Lab Investigation
3.1 to distinguish nucleotide differences between normal and mutant genes associated
with a selected disease.
In this unit, you will examine databases currently available based on molecular
sequencing of amino acids (proteins) and nucleotides (genes) and analyze similarities
and differences; as well as applications and significances to the study of origins.
PREPARATION
Materials and Equipment
A computer with internet access
Timing and Length of Labs
The Pre-lab has three parts: building structural and biochemical cladograms,
and analyzing cytochrome c similarities and differences among diverse taxonomic
groups. Some research is required in the first part and that could be done outside of
lab. Two lab periods, as a minimum, are required to complete the Pre-labs.
Part 1
This is a tutorial on using BLAST, which could be done together as a class or
individually. Screenshots are provided for each step of the way. A second lab period
could be used for discussion and analysis of results of the bioinformatics provided
by BLAST.
Guided inquiry starts with a tutorial (with step-by-step screenshots) on finding
a gene sequence in the Entrez Gene website; then students will need to do some
research for the name of the gene associated with the students selected disease.
They will then copy the gene sequence from the Entrez Gene website and paste it
into BLAST to compare the normal to the mutant gene sequence. This could take
two lab periods to research, complete and analyze.
Learning objectives aligned to standards and science

practices (SP):
To evaluate data-based evidence from bioinformatics tools like Entrez Gene
and BLAST to analyze evolutionary degrees of similarities and differences;
and to analyze genetic data related to diseases (1A2 and SP 5.3)
To construct a scientific explanation that uses the structures and mechanisms
of DNA and RNA to support the claim that DNA, and in some cases RNA,
is the primary source of heritable information (3A1 and SP 5.6)
To use cladograms and bioinformatics tools to ask other questions and test the
student's ability to apply learned concepts relating to genetics and evolution
(1A2. 1A4 and SP 1.1,1.2)
General Safety Precautions
There are no safety precautions associated with this investigation.
Pre-lab and Questions: Cladograms

What is a cladogram (CLAY-doe-gram)? It is a diagram that depicts the
probable evolutionary relationships among groups. It is based on phylogeny
which is the study of evolutionary relationships. Sometimes a cladogram is
called a phylogenetic tree. In the past, biologists would group organisms based
solely on their physical appearance. Today, with the advances in genetics and
biochemistry, biologists can look more closely at individuals in the hopes of
discovering patterns of evolution, and group them accordingly this strategy is
called evolutionary classification. Cladistics is a form of analysis that looks at
features of organisms that are considered "innovations," or newer features that serve
some kind of purpose. It is important to recognize that cladograms were created by
biologists to illustrate their understanding of progressive evolutionary development
over time; which is progressive evolution from varying taxonomic groups such as
evolution of reptiles to birds.
In this Pre-lab you will use the cladogram to illustrate group relationships
by their anatomical structures. Corresponding organs and other body parts that
are alike in basic structure and origin are said to be homologous structures (for
example, the front legs of a horse, wings of a bird, flippers of a whale, and the arms
of a person are all homologous to each other). Biological evolutionists have defined
homology as correspondence of structure derived from a common primitive origin.
This definition assumes progressive evolution over time from one taxonomic group
to another. Human hands and dog paws are an example of Darwinian homology.
Since the similarity of a human hand and a dog paw is somewhat structural, but not
functional, it is assumed that their similarity results from a common ancestor that
possessed this basic arrangement of bones. However, it should be noted that some
scientists interpretations of the actual embryonic origin of some supposedly
homologous structures is different.
Cladograms are used to show these assumed homologous structures. When
studies are done in comparative anatomy and different numbers of shared derived
characters are found to exist between different groups, a diagram can be created
with branching lines that connect those groups, showing their different degrees
of relationship. These diagrams look like trees. The organisms are at the tips of
the stems. The shared features of the homologous structures are shown on the
cladogram by solid square boxes along the branches, and predicted common
ancestors are shown by open circles. The more derived structures two organisms
share, the closer is their presumed evolutionary relationship that is, the more
likely their common ancestor lived recently. On the cladogram, closer predicted
relationships are shown by a recent fork from the supporting branch. The closer
the fork in the branch between two organisms, the closer is their relationship.
Procedures
Part A: Structural Cladogram
(Adapted from ENSI/SENSI lesson plan: Making Cladograms http://www.

indiana.edu/~ensiweb/home.html)
1. Using resources: internet, textbook, and the explanations below;
determine which of the characteristics each animal has. In the
Data Table provided on the next pages place an X in the box if
the animal has the characteristic.
Explanations of Characteristics:
Set #1:
Set #2:
Set #3:
Set #4:
Set #5:
Set #6:
Set #7:
Dorsal nerve cord: running along the back or dorsal

body surface.
Notochord: a flexible but supporting cartilage-like rod
running along the back or dorsal surface.
Paired appendages: legs, arms, wings, fins, flippers,

antennae.
Vertebral column: backbone.
Paired legs
Amnion: a membrane that holds in the amniotic fluid
surrounding the embryo; may or may not be inside an
egg shell.
Mammary glands: milk-secreting glands that nourish
the young.
Placenta: structure attached to inside of uterus of
mother, and joined to the embryo by the umbilical cord;
provides nourishment and oxygen to the embryo
Canine teeth short: same length as other teeth.
Foramen magnum forward: spinal cord opening, located

forward, under skull.
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Pre-lab Table 1
Animals
Kangaroo Lamprey Rhesus
Monkey
Sets
Traits
Set 1
Set 3
Dorsal Nerve Cord/

Notochord
Paired Appendages/
Vertebral Column
Paired Legs
Set 4
Amnion (Amniotic sac)
Set 5
Mammary Glands
Set 6
Placenta
Set 7
Canine teeth short/Foramen

magnum forward
Set 2
Bullfrog
Human
Snapping Tuna
Turtle
Total Number of X
2. Make a Venn diagram, placing your seven animals in groups to

illustrate those characteristics which different animals have in
common. See example below
3. Using the Venn diagram of the groupings just completed (as

a guide), draw a cladogram to illustrate the ancestry of these
animals. The diagram should reflect shared characteristics as
time proceeds. An example is
shown to the left. Notice how
the different animals are all at
the same time level (across the
top) since they all live today.
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Here is a fossilized walrus skull and tusks. This

demonstrates that not all fossils that we find represent
extinct organisms. Other examples like this include
clam shells, shark teeth, and bones.
Questions to consider
Are fossils only from extinct organisms?
Do fossils have to be thousands or millions of

years old?
How short of time can the fossilization

process take?
Part B: Biochemical Analysis for Cladogram Construction
GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is an enzyme that

catalyzes the sixth step in glycolysis, an important reaction in the process of cellular
respiration. The following data table shows the percentage similarity of this gene
and the protein it expresses in humans versus other species. For example, according
to the table below, the GAPDH gene in chimpanzees is 99.6% identical to the gene
found in humans.
Pre-lab Table 2
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Part C: Biochemical Analysis of Cytochrome c
Cytochrome c oxidase is another protein enzyme. It is involved in mitochondrial

electron transport at the later stages of ATP production. Cytochrome c differs in
every organism in its sequencing. The following Table 3 shows the percent of
difference in amino acid sequence in cytochrome c between 17 organisms. Reading
across the line marked humans, we see that the differences in sequence becomes
greater the farther away we move on the taxonomic scale. From human to Rhesus
monkey is only one percent divergence; from human to pig is ten percent; from
human to fish (carp) is 17%; from human to an insect is 29%. This finding is not
surprising since it corroborates traditional taxonomic categories.
Take a closer look at the silkworm, number 15 at the top of the table. As you
progress down the silkworm column through the different vertebrate classes, it
becomes apparent that the difference from diverse organisms is the same. It doesnt
matter if it is a human, a penguin, a turtle, fish, or lamprey. The silkworm differs
from all of these (reptiles, birds, mammals) by almost exactly the same percent.
Pre-lab Table 3
Comparisons of the Cytochrome c Molecule Sequencing % Differences of 17 Organisms
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Questions
Part A: Structural Cladogram
1. What are three types of information that can be obtained from

this cladogram?
2. Three previously unknown vertebrates have been discovered in

a rain forest in South America. One animal is very similar to an
iguana lizard. The second animal resembles a large rat. The third
is similar to a goldfish. Place these animals on your cladogram
and explain why you placed them where you did.
Part B: Biochemical Analysis for Cladogram Construction

3. Why is the percentage similarity in the gene always lower than
the percentage similarity in the protein for each of the species?
(Hint: Recall how a gene is expressed to produce a protein.)
4. Draw a cladogram depicting the evolutionary relationships

among all five species (including humans) according to their
percentage similarity in the GAPDH gene.
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5. What are the assumptions and evolutionary perspectives

associated with the cladogram?
Part C: Biochemical Analysis of Cytochrome c
Pre-lab Table 4: MODEL Data Chart

Position #12 Carp
Organism of
Organism of
(fish)
comparison Position # comparison
Carp (fish)
#10
Bullfrog (amphibian)
Cytochrome c from
organism of comparison
% Difference
13%
Carp (fish)
#9
Turtle (reptile)
13%
Carp (fish)
Chicken (bird)
Carp (fish)
Rabbit (mammal)
Carp (fish)
Horse (mammal)
6. Refer to the Cytochrome c Comparison Chart above. Carp (fish)
is in position #12. If you go to the Bullfrog amphibian, position
#10 and look across the horizontal line of numbers until you
get to the vertical column #12 (carp), you will find a difference
of 13% in the cytochrome c sequencing. Repeat for the turtle
(a reptile). Record the Cytochrome c % Difference for each
organism listed.
7. If fish evolved into amphibians, do you see molecular evidence

from cytochrome c sequencing % differences that fish are closer
to bullfrogs than to reptiles or mammals?
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8. How does this relate to what biological evolution might expect

if evolution from one taxonomic group to another occurred?
9. Does this evidence support or contradict the expectations of

Darwinian evolution?
10. Do you see any indication from the amino acid sequence

differences that there are transitional or intermediate species
from one taxonomic group to another?
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Part 1 - BLAST Practice
The figure above is a fossil cladogram.
BLAST Practice with an Unknown

Fossil Specimen
A team of scientists uncovered
the fossil specimen pictured at left in
China. DNA was extracted from a
small amount of soft tissue and the
sequence of nucleotides was determined
You will use the BLAST database to
analyze the sequencing and determine
the most likely placement of the fossil
species on fossil cladogram above.
Photo by Sam Ose & Olai Skjaervoy.
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Procedures
1. Form a hypothesis as to where you believe the fossil specimen
should be placed on the cladogram your observations.
2. Locate and download gene files. Download three gene files
from the following website http://blogging4biology.edublogs.
org/2010/08/28/college-board-lab-files/
Click on Gene 1 and this is what you see:
Click on Download and select Save File, OK
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3. Upload the gene sequence into BLAST by doing the following:
Go to the BLAST homepage
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Click on Saved Strategies from the menu at the top of the

page (see the red arrow 1 pointing to the highlighted area).
1. Saved
Strategies
button
2. Scientists
search within
several databases.
In this lab,
students will be
using nucleotide
blast to compare
the nucleotides
found in the
fossil specimen to
those in known
organisms.
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1. Click on
Browse and
then locate your
downloaded
Gene 1 file from
Biology website
and saved to your
computer.
2. Then click
View.
Under Upload Search Strategy, click on Browse and

locate Gene 1 files that you saved onto your computer (from
your download files).
Click View.
1. This is the
nucleotide
sequence found
in Gene 1. DO
NOT alter any
of the parameters
set on this page.
2. This setting
indicates the
entire database
will be searched.
Selecting the
human database
+ transcript
would only yield
similar sequences
found in humans.
By selecting
Others, all of
the genomes in
BLAST database
will be searched.
3. Select
BLAST
(Step 3e).
A screen will appear with the parameters for your query

already configured. NOTE: DO NOT alter any of the
parameters. Scroll down the page and click on the BLAST
button at the bottom.
After collecting and analyzing all of the data for that particular
gene (see instructions below), repeat this procedure for the
other two gene sequences.
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1. This bar
represents the
gene sequence
you entered into
BLAST.
2. This indicates
the number
of results (53
BLAST hits in
this example for
Gene 1).
4. The results page has two sections. The first section is a graphical
display of the matching sequences.
4
2
1
3. These bars
represent the top
results in the query
and how well
aligned each result
is with the gene of
interest.
4. This is the
Distance Tree of
Results button.
1. This is the
species and gene
name that matches
the gene of interest.
2. The score
(Max) refers to
how many gaps or
substitutions are
associated with
the sequence. The
higher the score,
the more similar
the alignment.
3. The e value is
the likelihood
that a match
occurred purely
by chance. The
lower the e value,
the more similar
the alignment (the
better the match).
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3
Scroll down to the section titled Sequences producing
significant alignments. The species in the list that
appears below this section are those with sequences
identical or most similar to the gene of interest. The
most similar sequences are listed first, an as you move
down the list, the sequences become less similar to your
gene of interest.
3
If you click on a particular species listed, youll get a

full report that includes the classification scheme of the
species, the research journal in which the gene was first
reported, and the sequence of bases that appear to align
with your gene of interest.
1. This indicates
the species the
aligned sequence
is found in
and the gene/
phenotype.
2. This describes
the number
of identical
nucleotides found
in this sequence.
3. This shows the
exact pattern on
alignment. The
top line is the
gene of interest
(Gene 1 in this
case) and the
bottom line is
the matching
sequence.
If you go back to the graphical (Distribution of BLAST

hits), click on the Distance Tree of Results, (4. Left
is pointing to the Distance Tree of Results button to
click) you will see a cladogram with the species with
similar sequences to your gene of interest placed on the
cladogram according to how closely their matched gene
aligns with your gene of interest.
Analyzing Results
5. After you have completed your data inquiry for all three
genes, you should be thinking about your original hypothesis
and whether the data support or cause you to reject your
original placement of the fossil species on the cladogram.
For each BLAST query, consider the following:
The higher the score, the closer the alignment.
The lower the e value, the closer the alignment.
Sequences with e values less that 1 X 10-4 can be

considered related with an error rate of less than 0.01%.
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Data Analysis and Conclusions

1. What species in the BLAST result has the most similar gene
sequence to the gene of interest?
2. Where is that species located on our cladogram?
3. How similar is that gene sequence?
4. What species has the next most similar gene sequence to the
gene of interest?
5. Based on what you have learned from the sequence analysis and
what you know from the structure, decide where the new fossil
species belongs on the cladogram with the other organisms and
redraw your original cladogram.
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In this guided inquiry, you will select a gene of interest, do a BLAST query on
your gene and determine the difference between the mutant disease causing gene
and the normal gene.
You will need one more skill before beginning this activity. In Lab 3.1, you
were provided with gene sequences from a database. This time, you will choose
your own gene to investigate so you will need to find your genes sequence in the
Entrez Gene database.
Following is a short practice activity with the gene for actin (a muscle protein
in humans that forms the microfilaments) that will guide you into the Entrez Gene
website for getting the nucleotide sequence for actin.
Tutorial on getting gene sequences:
1. Go to the Entrez Gene website http://www.ncbi.nlm.nih.gov/

gene and type human actin in the top search block.
2. This screenshot is the result of your search for human actin. Click
on the first link that appears.
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2. (Continued) Scroll down about two-thirds of the way to the

section NCBI Reference Sequences (in the upper left hand
corner of this screenshot).
3. Under mRNA and Proteins, click on the first file name. In this
case, it is under 1. NM_007353.2. It may be named something
similar depending on your gene of interest. These standardized
numbers make cataloging sequence files easier. Do not worry
about the file number for now.
4. Just below the gene title, click on FASTA. This is the name for
a particular format for displaying sequences.
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5. The nucleotide sequence displayed is that of the actin gene in

humans. The screenshot is not showing all of it it is important
in the next step to highlight and copy ALL of it for BLAST.
6. Copy the entire gene sequence, and then go to the BLAST

homepage http://blast.ncbi.nlm.nih.gov/Bast.cgi
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7. Click on nucleotide blast under the Basic BLAST menu.
8. Paste the entire gene sequence into the box where it says Enter
Query Sequence.
9. Under Choose Search Set, select whether you want to search

the human genome only or others.
10. Under Program Selection, choose whether or not you want

highly similar sequences (this would be best for a human disease
inquiry) or somewhat similar sequences if you want more results
in the case of an evolutionary relationship inquiry.
11. Click BLAST.
This ends the tutorial on finding your own gene sequences for BLAST.
Student Guided Inquiry Activity:
Identify a disease that is known to be related to proteins; identify the gene

involved; search for the normal sequence of the gene in the Entrez Gene website;
then upload it into BLAST. (The Entrez Gene website will only have the normal
gene sequence. Usually there are hundreds to thousands of different chromosomal
aberrations or mutations in the form of deletions, duplications, insertions, inversions,
or point mutations that will cause many of these diseases).
Using BLAST for the normal gene involved in the disease, research the mutant
versions of the disease and find out what is different about their sequences. Here
are some diseases to choose from:
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Genetic defects cause diseases in a variety of ways. The simplest way is through
a loss-of-function mutation. In this type of defect, a change in the DNA
nucleotides prevents the gene from making protein, or prevents the protein
from functioning once it is made. Genetic diseases due to loss-of-function
mutations are very common, and include cystic fibrosis (which affects the
lungs and pancreas), Duchenne muscular dystrophy, and the hemophilias, a
group of blood clotting disorders.
A second mechanism for causing disease is called a toxic-gain-of-function
mutation. In this type of defect, the gene takes on a new function that is
harmful to the organismthe protein produced may interfere with cell
functions, or may no longer be controllable by its normal regulatory partners,
for instance. Many degenerative diseases of the brain are due to this type of
mutation, including Huntington's disease.
Read more: http://www.biologyreference.com/Fo-Gr/Genetic-Diseases.
html#ixzz2MUxHudP4
You will need to research what gene is associated with your selected disease
before going to the Entrez Gene website.
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Data Analysis and Conclusions

1. What were your findings in comparing normal to mutant gene
sequences of your selected disease?
2. How can this help in treating or preventing this disease?
3. What are scientists doing today in genetic engineering with the

knowledge of this gene deficiency?
28

AP Biology Lab-03

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Lab 3

Comparing DNA Sequences to Understand

How can bioinformatics be used as a tool to determine evolutionary

Please be sure you have read the

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations Summary

Part A - Structural Cladogram

Lab Investigation 3.1

Part 1 - BLAST Practice

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LAB 3 - Comparing DNA Sequences to

Consider how (or if) a multitude of testable hypotheses can be applied

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changes in the genetic material of different taxonomic groups of organisms and

Are similarities in biochemical molecules evidence of evolution? For

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fish (carp). Another puzzle to be solved.

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Timing and Length of Labs

Lab Investigation 3.1

Learning objectives aligned to standards and science

General Safety Precautions

There are no safety precautions associated with this investigation.

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Pre-lab and Questions: Cladograms

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(Adapted from ENSI/SENSI lesson plan: Making Cladograms http://www.

Dorsal nerve cord: running along the back or dorsal

Paired appendages: legs, arms, wings, fins, flippers,

Foramen magnum forward: spinal cord opening, located

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Dorsal Nerve Cord/

Amnion (Amniotic sac)

Canine teeth short/Foramen

2. Make a Venn diagram, placing your seven animals in groups to

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3. Using the Venn diagram of the groupings just completed (as

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Here is a fossilized walrus skull and tusks. This

Do fossils have to be thousands or millions of

How short of time can the fossilization

Part B: Biochemical Analysis for Cladogram Construction

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is an enzyme that

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Part C: Biochemical Analysis of Cytochrome c

Cytochrome c oxidase is another protein enzyme. It is involved in mitochondrial

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1. What are three types of information that can be obtained from

2. Three previously unknown vertebrates have been discovered in

Part B: Biochemical Analysis for Cladogram Construction

4. Draw a cladogram depicting the evolutionary relationships

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5. What are the assumptions and evolutionary perspectives

Part C: Biochemical Analysis of Cytochrome c

Pre-lab Table 4: MODEL Data Chart

7. If fish evolved into amphibians, do you see molecular evidence

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8. How does this relate to what biological evolution might expect

9. Does this evidence support or contradict the expectations of

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Lab Investigation 3.1

The figure above is a fossil cladogram.

BLAST Practice with an Unknown

Photo by Sam Ose & Olai Skjaervoy.