Biology 212 Genetics Lab
Spring 2007
Lab 1: DNA Models
Hemoglobin Electrophoresis
Purpose:
1. Be able to identify the building blocks of DNA and RNA.
2. To assemble a strand of DNA and copy it into RNA.
3. To build a polypeptide chain (protein).
4. Associate the structure of a protein (hemoglobin) with its function.
5. Identify mutations as a cause of variability and genetic disease
Reference: Jones and Hartl, Chap. 1
Background on DNARNAprotein 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:
PPPPPP
SSSSSS
||||||
TGGACG
ACCTGC
||||||
SSSSSS
PPPPP 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.
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:
--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.
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.
Lane 1 / Lane 2 / Lane 3Normal hemoglobin / Sickle cell
hemoglobin / Carrier
sample
15 ul / 15 ul / 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).
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:
3. Give a one or two sentence overview of what happens in the following processes. Thenname key enzymesor components and their roles in each process.
a. REPLICATION--
Key enzymes/components:
b. TRANSCRIPTION—
Key enzymes/components:
c. TRANSLATION--
Key enzymes/components:
4.a. Draw a representation of the hemoglobin electrophoresis gel pattern in the space below. Label the lanes and their contents.
Sample lane1 2 3
Sample contents
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 cells6. 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 codon 5 codon 6 codon 7 codon 8
DNA / 5’ C / C / T / G / A / G / G / A / G / A / A / G 3’DNA / 3’ G / G / A / C / T / C / C / T / C / T / T / C 5’
mRNA / 5’C / C / U
Protein NH2COOH
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 codon 5 codon 6 codon 7 codon 8
DNA / 5’ C / C / T / G / T / G / G / A / G / A / A / G 3’DNA / 3’ / 5’
mRNA / 5’
Protein NH2COOH
1