Download Pre-lab Homework Lab 3: DNA Structure and Function

Biology 102
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Name: ______________________________________
Pre-lab Homework Lab 3: DNA Structure and Function
1. Draw and define the following in your own words. Using your textbook, or any other resource, is
ok; just rephrase the definitions in your own words!
•
DNA:
•
Deoxyribose:
•
Nucleotide:
2. What is the complementary base pairing for the following (DNA and RNA)?
DNA 5’-A T C T A G C A T-3’
DNA 5’-A T C T A G C A T-3’
Complementary DNA
Complementary RNA
3. The four nucleotide monomers of DNA are made up of 3 smaller molecules joined together. These 3
smaller molecules - a phosphate group, a deoxyribose sugar, and a nitrogenous base - are covalently
bound together to build each nucleotide.
A. Draw one nucleotide and label each of the 3 parts. You don’t need to label all the atoms; just show
the basic structure of the monomer.
B. Indicate the 5’ and 3’ carbons that covalently link one nucleotide to the next.
4. What are 2 structural differences between DNA and RNA?
5. How does DNA store genetic information (i.e. the information to build proteins)?
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Name: _______________________________________
Date/Lab time: ___________________
Lab 3: DNA Structure and Function
LAB SYNOPSIS:
• DNA replication will be modeled using nucleotide cutouts.
• Transcription of DNA to RNA will be modeled using nucleotide cutouts.
• Translation of mRNA to an amino acid sequence will also be modeled.
• Gel electrophoresis process will be done using colored dyes.
• DNA fingerprinting will be described using a scenario.
OBJECTIVES: After successfully completing this lab, a student will be able to:
• Describe the structure of DNA and how this structure allows replication.
• Demonstrate the processes of DNA replication, transcription & translation
• Explain how changes in DNA structure can cause changes in protein structure.
Overview:
DNA (deoxyribonucleic acid)- A long nucleic acid polymer made up of nucleotide
Figure 1. DNA
monomers.
Function- Carrier of genetic information. Codes for the genetic characteristics that make you.
Structure- Double helix (two twisted strands held together by hydrogen-bonds) (Fig. 1 & 2).
Chromosome- Organized structures of DNA and protein. Become visible during mitosis.
The building blocks of DNA are 4 nucleotides (adenine, guanine, cytosine & thymine)
Label each on figure 2:
• Nucleotides, each is made up of 3 parts.
o Deoxyribose (a sugar containing 5 carbons)
o Phosphate group (linked to the deoxyribose 5’
carbon)
o Nitrogenous base (either A, T, C or G)
•
Sugar-Phosphate backbone. Each of the two
strands is held together by strong covalent bonds
between the 5’ carbon of one deoxyribose and the
3’ carbon of the next deoxyribose.
o 5’ to 3’ is the direction the DNA is synthesized.
•
The two strands of DNA run antiparallel to one
another. Note the arrows.
•
The two strands of DNA are held together via H-bonds.
Note the dotted lines between.
o Complementary base pair rules.
T base pairs with A
G base pairs with C
Be able to identify the phosphate group, deoxyribose sugar and the 2
strands.
Figure 2. DNA Structure
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Exercise 1: DNA Replication. Modeling.
DNA replication- the process of making an exact copy of each of the two strands of DNA.
DNA unzips. Each stand acts as a template for the construction of a new complementary strand of
DNA (Fig. 1). We will model the structure of DNA using basic cardboard cutout pieces to representing
the nucleotide monomers of DNA. These pieces represent the sugar, phosphate, and nitrogenous
base of a nucleotide and come in four different versions to represent each of the four different types of
nucleotides (G, A, T, & C).
Procedure:
1. Obtain and sort a DNA modeling set. Separate the
pieces into stacks representing each of the four types of
nucleotides (Fig. 3). The pieces labeled as tRNA and
amino acids should be set aside; we will use them later.
2. Once you have sorted your nucleotides, build a chain of
nucleotides that reads:
5’-G-A-T-T-A-C-G-C-C-G-A-C-3’
Figure 3. Four Nucleotide Model Pieces
This represents one strand of the DNA molecule.
3. Now build the other strand of your DNA molecule by following the complementary base pairing
rules of A pairing with T & G pairing with C. Remember: the strands run antiparallel to each other.
4. Your final model now represents a very short molecule of double-stranded DNA. (note: the shortest
human chromosome contains about 50 million nucleotides!)
DNA Replication:
Now replicate the chromosome you have constructed.
5. To do this, split the two strands in half by separating the base pairs. Use each of strand of
nucleotides as a template for a new strand by once again following the complementary base pair
rules (A-T & G-C).
6. Compare the resulting molecules to each other. They should be identical.
What will the sequence be for the complementary strand to?
5’-G-A-T-T-A-C-G-C-C-G-A-C-3’
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Exercise 2: Transcription of DNA into mRNA, Modeling
In this exercise, you will use your DNA model from Exercise 1 to serve as a template for making a
mRNA transcript, and then will use tRNA molecules to decode the information on the mRNA and construct
a short chain of amino acids. Transcription happens in the nucleus of eukaryotic cells, while translation
occurs on ribosomes.
Procedure:
1. Start with one of the double-stranded molecules of DNA you created in Exercise 1 (the other molecule
you can “digest” into nucleotides for use in creating your mRNA). This double-stranded DNA should
have the sequence:
5’-G-A-T-T-A-C-G-C-C-G-A-C-3’
: : : : : : : : : : : :
3’-C-T-A-A-T-G-C-G-G-C-T-G-5’
2. Now pull apart the two halves of your DNA molecule. One strand of this molecule will be copied into
mRNA (we call this the sense strand) while the other strand is not used (we call this the anti-sense
strand).
3. RNA is synthesized in a 5’to 3’ direction. So we will copy the bottom DNA strand that starts with 3’- CT-A-A-T….. Remember that in RNA molecules, the letter T is replaced by the letter U- so the base
pairing rules are A-U, T-A, C-G, G-C.
Write down the sequence of your mRNA that is complementary to the following DNA;
3’-C-T-A-A-T-G-C-G-G-C-T-G-5’
4. This mRNA molecule now represents a set of instructions for the order of amino acids needed for a
protein. Translation is the process of “decoding” the information of mRNA into a sequence of amino
acids.
Exercise 3: Translation of mRNA into a Sequence of
Amino Acids, Modeling
The models do not work well for the process of translation.
1. Use the genetic code chart to determine the amino acid
sequence your mRNA codes for.
Write down the sequence of your amino acids.
How would the amino acid sequence change if a single
DNA point mutation occurred? How might this effect
protein structure and thus its function?
3’-C-T-A-A-T-G-C-G-G-C-T-G-5’ 3’-C-A-A-A-T-G-C-G-G-C-T-G-5’
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Exercise 4: Strawberry DNA Extraction
Although we cannot see individual molecules of DNA at the molecular level, we can isolate large
quantities of DNA by breaking open large numbers of cells. The goal of this laboratory exercise is for
you to see, touch and feel DNA. Scientists in biotechnology and research laboratories perform
essentially the same technique you are performing today to purify and study DNA.
How This Procedure Works:
1. Mashing: this process breaks the cell walls of the strawberry.
2. Extraction solution: this solution contains detergent, salt, and water.
a. The detergent breaks down membranes.
What membranes need to be broken to release DNA?
b. DNA carries a negative charge (see phosphate groups in fig. 2). DNA is therefore very soluble in
water, so when the cells are broken open, the DNA goes into solution.
c. The salt (NaCl) breaks down into ions in the water. Positively-charged sodium ions (Na+) are
attracted to and bind the negatively charged DNA molecules. This makes the DNA molecules
neutral, so the DNA clumps together.
3. Ethanol: Very cold alcohol acts like a lipid. DNA is not soluble in lipids. When cold ethanol is
added to the solution, the DNA can no longer stay in solution and forms a precipitate.
Procedure:
1. Gather all the materials you will need (except ice cold ethanol) including gloves and goggles, and
read through all of this procedure before beginning.
2. Remove the leaves from the top of strawberry and place fruit in re-sealable plastic bag.
3. Seal sandwich bag and squash the fruit with your palm. Squash as thoroughly as possible without
breaking the bag.
4. Add 10 mL of extraction solution to the bag with the pulverized fruit. Continue to squash mixture
for an additional two minutes.
5. Place a filter in the funnel and suspend funnel over glass beaker.
6. Pour the contents of the plastic bag into the filter and set aside for 10 minutes.
7. Discard filter and contents.
8. Pour the filtrate into a test tube.
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9. You now need cold ethanol. SLOWLY pour the cold ethanol down the side of the test
tube with the strawberry filtrate. The ethanol should form a layer onto the strawberry
filtrate. The DNA will start precipitating out in the alcohol almost immediately, but you
will want to gently put your test tube in the rack and wait a few minutes to get the
maximum effect. DO NOT MIX THE CONTENTS. You want the ethanol to remain a
distinct layer on the top of the filtrate.
10. Use the end of the Pasteur pipette (or glass rod) to carefully spool the precipitated DNA out of
the ethanol.
11. CLEAN up all material used as directed in lab. Glass rods can be washed and returned to the
equipment tray.
Some of the things that can be done with extracted DNA include: DNA sequencing, DNAfingerprinting, cloning, determining phylogeny, determining genetic disorder, determining cancer genes.
Rosalind Franklin used purified DNA to determine its X-ray diffraction pattern (used by Watson and
Crick to determine DNA’s structure).
Questions:
1. How would the DNA of strawberries differ from your DNA?
2.
How would the DNA of strawberries be similar to your DNA?
3. Your precipitated DNA contains both nucleic acids and proteins. What is the source of the proteins
in the strawberry extract? Hint: think about how DNA is arranged in a chromosome.
4. The nucleus of every human cell contains approximately 2 meters of DNA. A typical adult human
contains 20 trillion cells (20,000,000,000,000). The distance from the Earth to the Moon is 380,000
km. If all your DNA were laid end to end, how far would it reach? (you will need to convert meters
to kilometers)
4. Is there DNA in the food you eat? ________ Why do you think so?
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Exercise 5: Gel Electrophoresis
Gel Electrophoresis- a technique used for the separation of molecules using an electric field applied to
a gel matrix. Molecules can be separated based on their size and their electrical charge.
Gel electrophoresis is a commonly used technique in modern biology. It relies on the fact that
charged molecules will be attracted to oppositely-charged electrical current, and that small molecules
can move more quickly through complex polymers than can large molecules. There are different gel
electrophoresis methods used for differing purposes.
To demonstrate the use of gel electrophoresis, we will use it to separate different dyes.
Procedure:
1. Locate the agar gel rig on the prep counter. Agar is a polysaccharide extracted from seaweed. Dried
agar is mixed with hot water and forms a gelatinous matrix when cooled. The gel has small
chambers called wells in the middle (Fig. 4). You will load samples of dyes into the wells. The
various molecules that make up the dyes will then separate based on their size and electrical charge.
Figure 4: Gel set-up
The gel has wells down the middle where
you load samples. These samples migrate
toward poles with a charge that is the
opposite of the sample. Smaller molecules
move faster than larger ones. All like
molecules move at the same rate forming a
band.
2. Load the gel wells using the micro-pipette. Your group will be assigned one known molecule and
one unknown mix of molecules to load.
3. Once all 5 wells have been loaded, the gel will be submersed in a fluid that conducts electricity.
Once turned on, an electrical current will run through the gel. *Caution: high voltage* Never
touch a running gel rig!
Results:
After running the gel, results of gel electrophoresis will form discrete “bands” indicating separated dyes
of the same type of dye molecule.
Conclusion: Based on comparison with the bands of the known dye solutions;
Unknown Sample label Contents (dye(s) and their electric charge)
A
B
C
D
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Exercise 6: DNA Fingerprint Interpretation
DNA Fingerprinting- A technique comparing fragments of DNA separated by gel electrophoresis. The
resulting bands of separated DNA are unique to each individual and thus can be used for identification.
Principle: DNA from an individual is extracted and cleaved into pieces of differing sizes with
restriction enzymes. Restriction enzymes only cut at specific base sequences. If you cut the DNA in a
region that is highly variable, then different people will likely give different DNA banding patterns
following DNA gel electrophoresis (Fig. 5).
Figure 5. DNA Fingerprinting Protocol
Scenario:
You are a forensic biologist in a small town. Someone has been sneaking into the local grocery store
and stealing lollypops. The thief always eats a lollypop at the store and leaves behind the stick. The stick
will be covered with saliva and many cells from the thief’s mouth. Using various techniques you are
able to isolate DNA off these sticks, cleave the DNA with restriction enzymes and run a gel along side
DNA samples from 5 suspects in the lanes labeled A – E (Fig. 6). Note: DNA is negatively charge and
will separate based on size.
1. After examining the results, you call the police chief. What do you tell her?
The police chief then brings you a new set of DNA samples taken
from some of the criminals that she has in custody for other crimes
(Fig. 7).
Figure 6. DNA Fingerprinting, Run #1
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2. After examining these results, you call the police chief. What do
you tell her?
Figure 7. DNA Fingerprinting, Run #2
3. In 1998, Foster et al. used DNA fingerprinting similar to this to compare the DNA from people
known to be related to Thomas Jefferson to the DNA of people descended from one of Jefferson’s
slaves named Sally Hemmings. The information generated led the scientists to conclude that one of
Sally’s sons had been fathered by Jefferson, but that her other son had not. This study was
complicated by the thought that perhaps one of Jefferson’s nephews had been both boys’ father.
Why would it be more difficult to use this sort of information to distinguish between people who are
closely related?
Work cited:
Foster, EA; Jobling MA, Taylor PG, Donnelly P, de Knijff P, Mieremet R, Zerjal T, Tyler-Smith C
(1998). “Jefferson fathered slave's last child”. Nature 396 (6706): 27–28.
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