Download Lab 1

Biology 212 Genetics Lab
Spring 2007
Lab 1: DNA Models
Hemoglobin Electrophoresis
Purpose:
1.
2.
3.
4.
5.
Be able to identify the building blocks of DNA and RNA.
To assemble a strand of DNA and copy it into RNA.
To build a polypeptide chain (protein).
Associate the structure of a protein (hemoglobin) with its function.
Identify mutations as a cause of variability and genetic disease
Reference: Jones and Hartl, Chap. 1
Background on DNARNAprotein models:
Genes consist of DNA (deoxyribonucleic acid). DNA is made of the sugar,
deoxyribose, phosphate groups and four different bases, adenine (A), cytosine (C),
guanine (G) and thymine (T).
The structure of DNA consists of a chain of alternating sugars (S) and phosphates
(P); the bases form the rungs of the ladder:
P
P
P
P
P
P
S
|
T
S
|
G
S
|
G
S
|
A
S
|
C
S
|
G
A
|
S
C
|
S
C
|
S
T
|
S
G
|
S
C
|
S
P
P
P
P
P
P
Genes carry the information to make the proteins. Proteins determine most of our
traits. The sequence of bases on the DNA (CGATAC..) specifies the code for the
proteins. Proteins are made up of smaller units called amino acids.
To make a protein from a gene involves:
1. TRANSCRIPTION: The DNA containing the gene is copied into another
nucleic acid, messenger RNA. RNA is like DNA except it contains ribose as the sugar, it
has the base uracil in place of thymine and it is always a single strand.
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2. Messenger RNA moves to the ribosome, the site of protein synthesis.
3. TRANSLATION:
A. Transfer RNAs, small RNAs that decode the message, bring the amino acids
to the ribosome.
B. The transfer RNAs bind to the messenger RNA. A code of three bases (a
codon) is read by each transfer RNA.
C. Protein synthesis occurs when the amino acids carried by the transfer RNAs
are joined together by the ribosome to make a polypeptide chain (protein).
Procedure:
Models of DNA, RNA and proteins
In this part of the lab, you will use the DNA puzzle to build a DNA, transcribe it
into messenger RNA and start to build a protein. Work in small groups of 2-4 students
on the models as the kits become available. Note that you may want to use part of each
model in the next one you build.
DNA model
1. Sort out the building blocks for DNA from the other pieces. These are:
--deoxyribose sugars (salmon)
--phosphates (yellow)
--bases (green, blue, blue-green)
2. Build a backbone for the DNA from alternating sugars and phosphates.
3. Attach the bases to the sugars.
4. Show how the strand of DNA can be copied into another strand (this process is called
REPLICATION). Use the following base pairing rules
A pairs with T
G pairs with C
messenger RNA model
1. Locate the building blocks unique to RNA. These are:
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--ribose sugars (pink)
--uracil (U) base (white)
These are combined with phosphates (yellow) and the other bases to make RNA.
2. Construct a messenger RNA molecule that could be made from one of the DNA
strands you made. The same base pairing rules apply for RNA except uracil (U) pairs
with adenine (A).
Synthesize a protein
1. Identify the special pieces for protein synthesis in the kit. These are:
--the ribosome (large white folded sheet)
--transfer RNAs (large brown or tan cloverleaf)
--amino acids and charging enzymes (brown or tan tiles)
2. Link up the transfer RNAs with the correct amino acids and charging enzymes.
3. Bring together the different components for protein synthesis.
A. Open out the ribosome sheet
B. Move the messenger RNA to its place on the ribosome
C. Find transfer RNAs that can bind two sets of codons on the messenger RNA (you may
need to substitute for some of the bases on your messenger RNA as the kit can only
decode two of the possible twenty amino acids).
D. Link together the amino acids on the tRNA in the “A” site to start the protein chain.
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Background on Sickle Cell Anemia (Source: Ward's Natural Science)
Sickle cell anemia is a hereditary blood disease due to a defect in the hemoglobin
protein structure. The hemoglobin in people with sickle cell anemia differs from normal
hemoglobin at a single amino acid. Normal hemoglobin (HbA) consists of two alpha
polypeptide chains and two beta chains. The substitution of valine for glutamic acid in
the beta chain causes sickle cell anemia. This amino acid change induces a structural
change, causing the protein to precipitate and deform the red blood cell, causing the
sickle shape. The sickle cell beta chain is less able to carry oxygen, leading to symptoms
of anemia.
Sickle cell anemia is most common among persons of African descent, but is also
found in people from the Mediterranean and India. The sickle cell mutation is common
in regions where malaria, a frequently fatal blood disease caused by a parasite spread by
mosquitos, is epidemic. People with the sickle cell mutation are more resistant to the fatal
complications of malaria.
Electrophoresis has been used since 1949 to analyze the physical and chemical
properties of hemoglobin. In sickle cell hemoglobin (HbS), the presence of the amino
acid valine in place of glutamic acid changes its electric charge; it migrates more slowly
than normal hemoglobin. Heterozygous individuals are usually asymptomatic, but
display both types of beta globin chains in their blood; they are carriers of the sickle cell
trait. Today's lab will demonstrate the various hemoglobin electrophoresis patterns of the
different phenotypes.
Materials
Hemoglobin samples for normal, sickle cell, unknown
Micropipets, P20 or P200
Disposable yellow micropipet tips
Agarose gel
Electrophoresis chamber
Power supply
Slides of normal blood and sickle cell blood
Light microscopes
Procedure: Protein test for sickle cell anemia
work in groups of 4-6 students as indicated by the instructor
1. The electrophoresis chamber should be set up with the 1.3% (w/v) agarose gel (comb
removed) covered with electrophoresis buffer (1x Tris glycine buffer).
2. Set the micropipet to 15 microliters. Using a separate disposable tip for each sample,
transfer 15 microliters of each into the appropriate well. Dispose of tips in orange
biohazard bags provided.
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Lane 1
Normal
hemoglobin
15 ul
Lane 2
Sickle cell
hemoglobin
15 ul
Lane 3
Carrier
sample
15 ul
3. Attach the lid to the chamber. Attach the red and black leads to the power supply. Red
leads should be attached to the unit furthest from the sample wells.
4. Perform the electrophoresis at 100 V for about 1 hour or until the loading dye is at
least half to 2/3 of the way down the gel. If your gel is still running, observe the
demonstration gel at the front of the room before you leave.
5. Observe the gel and draw a replica of the protein separation pattern on the worksheet.
Please do not remove gels from units as they need to be decontaminated with bleach
solution.
Procedure: Blood cell morphology test for sickle cell anemia
1. At work stations, students should set up two microscopes side by side, one with each
type of blood slide (normal blood smear and sickle cell blood smear).
2. Observe the normal blood smear and sickle cell anemia blood smear slides under the
light microscope, on the second highest power. See your instructor if you need assistance
with the microscope. The highest power can be used, but you will need to add immersion
oil to the slide, supervised by the instructor.
2. Make pencil drawings of the red blood cells and include in the spaces provided on the
worksheet. Give the magnification of your drawing (ocular lens x objective lens).
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Name____________________________
Section____________________
Bio 212 DNA Lab Assignment (20 points): Answer the following questions and
hand in by next week’s lab.
1. a. What are the chemical building blocks of DNA?
of RNA?
of proteins
2. What is the function of
a. DNA:
b. messenger RNA:
c. transfer RNA:
d. proteins:
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3. Give a one or two sentence overview of what happens in the following processes.
Then name key enzymes or components and their roles in each process.
a. REPLICATION--
Key enzymes/components:
b. TRANSCRIPTION—
Key enzymes/components:
c. TRANSLATION--
Key enzymes/components:
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4.a. Draw a representation of the hemoglobin electrophoresis gel pattern in the space
below. Label the lanes and their contents.
Sample lane
Sample contents
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2
3
b. Based on the pattern of mobility (recall that normal hemoglobin migrates more
quickly), identify the protein bands for the normal, sickle cell and carrier individuals on
the gel diagram above. These are naturally rust red in color, while the loading dye is
blue.
5. In the boxes below, provide drawings of normal red blood cells and blood cells from a
patient with sickle cell anemia:
Normal red blood cells
Sickle cell red blood cells
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6. a. What is a mutation?
b. What is the relationship between a mutation on the DNA and the structure and
function of a protein, such as hemoglobin?
7. The normal DNA sequence of the B-globin gene (encoding one of the subunits of
the hemoglobin protein) is:
normal
DNA
5’ C
DNA
3’ G
mRNA 5’C
codon 5
C
G
C
codon 6
T
A
U
G
C
A
T
codon 7
G
C
G
C
A
T
codon 8
G
C
A
T
A
T
Protein NH2
G 3’
C 5’
COOH
In sickle cell anemia, the DNA sequence has a single point mutation (italics).
Complete the tables, giving the DNA, RNA, and protein sequences for the normal
and sickle cell versions
sickle cell
DNA
5’ C
DNA
3’
mRNA 5’
codon 5
C
T
codon 6
G
T
G
Protein NH2
codon 7
G
A
codon 8
G
A
A
G 3’
5’
COOH
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