Fundamentals of Chemistry 1030

Nucleic Acids

Purpose:

To make a model of DNA, determine how it codes for a protein and see the effect of a mutation.

To extract the DNA from a piece of fresh onion.

Introduction:

DNA (deoxyribonucleic acid) is the intriguing molecule that determines the basic structure and function of living things. It is the genetic material that allows offspring to be like their parents whether the parent is a bacterium or a chimpanzee or a pine tree of a human. If this molecule changes, the organism is apt to change also. This exercise will focus on this molecule -- what it looks like, what it does and what happens when it changes.

DNA and RNA are the nucleic acids found in the nuclei of cells. Both are made up of many nucleotides linked together.

A nucleotide consists of a nitrogen base, a sugar (deoxyribose or ribose) and a phosphate group. You should be able to identify these components of a nucleotide.

DNA (deoxyribonucleic acid)

Nucleotides in DNA have one of four nitrogen bases: adenine, thymine, guanine and cytosine.
The sugar in DNA is called deoxyribose because it does not have a hydroxyl group bonded to the 2' carbon.

RNA (ribonucleic acid)

Nucleotides in RNA have one of four nitrogen bases: adenine, uracil, guanine and cytosine.
The sugar in RNA is called ribose and it does have a hydroxyl group bonded to the 2' carbon.

In both DNA and RNA, nucleotides are bonded together to form strands of nucleic acids. The nucleotides are linked by the sugar of one nucleotide to the phosphate group of a different nucleotide. These linkages are called phosphate linkages and they make up the backbone of the nucleic acid strand. A template of a single strand of nucleic acids is shown in Figure 1.

Figure 1. Basic structure of a single strand of nucleic acids.

In DNA, two nucleotide strands interact to form a double strand as shown in Figure 2. These interactions occur between specific bases: adenine base-pairs with thymine and guanine base-pairs with cytosine.

Figure 2. Basic structure of a double strand of nucleic acids showing base-pairing.

Activities:

Part I.

Isolation of DNA from an Onion

1. Place a piece of onion (about the size of a quarter in a 100-ml beaker with 10 ml of the detergent/salt solution. Macerate the onion with the end of a scoopla by pressing it up against the side of the beaker.

2. Decant the liquid from the mixture into a clean test tube. Add 3 to 4 drops of meat tenderizer/enzyme solution. Swirl test tube to mix.

3. Carefully pour 10 ml of ice cold ethanol down the side of the test tube to form a layer on top of the onion mixture. Let stand 3 minutes.

4. Using a twirling motion of the glass rod or rubber tip, slowly move the end of a glass rod through the interface of the two layers to collect the mucus-like DNA and place on a watch glass to dry. You may wish to place the watch glass in a 95 degree oven for 15-30 minutes to facilitate the drying process. What does the DNA look like?

Part II:

A. Model of DNA

1) Obtain a bag of beads. The color of the bead represents its structure as follows:

White - sugar

Red - phosphate

Orange - adenine (A)

Green - thymine (T)

Yellow - guanine (G)

Blue - cytosine (C)

Pink - uracil (U)

Clear connectors - hydrogen bonds between base pairs

2) Make a chain of alternating red and white beads until you have a chain that is 20 beads long. This represents the backbone of one strand of DNA.

3) Attach orange, green, yellow or blue beads to each white bead in your DNA backbone in any order you choose. This is a model of a single strand of DNA made up of a chain of nucleotides.

4) Using the clear connectors, attach colored beads to each orange, green, yellow and blue bead in your strand using the following pattern:

Green connects to orange

Orange connects to green

Blue connects to yellow

Yellow connects to blue

5) Attach a white bead to the colored beads you have just added. Connect a red bead between each white bead. You now have a model of a double strand piece of DNA. Draw your piece of DNA in the Data/Observations Section. Use Figure 2 as a model for your drawing, but insert the name of the bases from you model instead of the word "base".

6) Hold your double strand piece of DNA by each end. Twist it one full turn. You have now made a model of a piece of DNA just as you would find it in your cells - a double stranded helix.

B. DNA Replication

Whenever a cell divides to make a new cell, either for new organism of for growth and repair of an individual, more DNA must be made. The method used allows for copies with the fewest possible errors. Try it!

1) Straighten the helix that you made in the activity above.

2) Partially separate the two strands by pulling apart the connectors between the first 5 base pairs starting from either end. (separate the green from the orange, etc.)

3) To the unpaired bases, attach the complementary bead to each green, orange, blue and yellow bead following the pattern used before (note: you will also need to add connectors - "hydrogen bonds" to one half of the strand. Attach a white bead to the colored beads as you did before and connect a red bead between each white bead. Make a sketch of the DNA replication process that you have modeled in the Data/Observations Section.

If you were to continue separating your original piece of DNA and add the complimentary strand, you would then have two pieces of DNA just like the original. Each piece would have one old strand and one new strand. This method of copying is called semiconservative replication. What is the advantage of that kind of replication? (Answer in Data/Observations Section)

C. Protein Synthesis

An important task of DNA is to provide the pattern or recipe for the structure and activity of a cell. It does this by making molecules called proteins. Proteins are made up of amino acids just as a necklace is made up of beads. The order in which you place the beads determines what the necklace looks like. The order of amino acids in a protein determines what the protein looks like as well as how it functions. The order of the nucleotides (A, T, C, & G) in the DNA codes for or determines the order of the amino acids in the protein.

While DNA is responsible for the amino acid sequence, it does not actually do the construction of the protein itself. That is where RNA comes into play. RNA is very similar to a single strand of DNA. It has a different sugar (ribose) and uracil (U) instead of thymine (T). The U acts just like a T and matches up with an A. Every three bases (ACC, CGA, etc.) are the code, or codon, for one amino acid as shown in the chart below. If a codon encodes for a STOP, then that is the end of the protein.

Use the following activity to get the idea…

1) Using your model from the previous activity, break all of the hydrogen bonds (take apart the connectors) and choose a single strand of DNA.

2) Remove one nucleotide from the end so that your strand of DNA has 9 bases.

3) Attach the colored beads just as you did earlier in the DNA exercises, except use pink instead of green. Match pink to orange, orange to pink, blue to yellow, and yellow to blue. You have now made a strand of RNA. It is called messenger RNA because it carries the message from the DNA.

4) Translate the bead colors in the new strand (your RNA) into the base letters, A, U, G, C and write them down below. Use the color code: orange = A, pink = U, yellow = G, and blue = C.

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5) Divide the letter you have recorded above into groups of three beginning from the left i.e., ACU/CGC/AUC

6) Using the attached chart, find the amino acids that make up your protein and record it on the line.

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For example: AUG

Find the first base A on the left side.

Next, find the second base U on the top

Then, find the third base G within that block.

AUG encodes for met which is the abbreviation for the amino acid methionine.

D. Mutations

A mutation is any permanent change in DNA so any new cell gets the change and the new cell follows the new directions. Usually, that doesn't do good things for the cell as the old plans worked pretty well. However, it might just be a change that allows an organism to adapt to a new environment such as a bacteria that causes pneumonia being able to live in the presence of an antibiotic -- not good for you, but good for the bacteria. Cells do have good repair systems to keep making the same DNA over and over again, but when these systems fail, you have a mutation. Radiation, toxic chemicals and old tired organisms lead to mutations.

What happens in a mutation? There are a number of possibilities. An entire piece of DNA can be destroyed which means that the blueprints are gone. No blueprints -- no protein --means trouble. What about other changes where the blueprint (DNA) has been altered but not destroyed? See what happens in the following exercise.

1) Using the following sequence of DNA, write out the strand of RNA that would be produced. (remember, there will be no T in your RNA)

DNA:

RNA:

2) Divide the letters into groups of three beginning at the left as you did earlier. This gives you your codons.

3) Using the code chart, determine the amino acid sequence in the protein that this RNA produces.

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4) Rewrite the original piece of DNA from the table above, but change one of the bases somewhere in the strand and then repeat the exercise. What happens to your amino acid sequence? Did it change?

DNA:

RNA:

Data/Observations Section

Part I.

What did the isolated onion DNA look like before it was dried?

What did it look like after it was dried?

Part II.

Section A, #5. Draw your piece of DNA.

Section B, # 3a. Sketch the DNA replication process that you have modeled.

Section B # 3b . What is the advantage of semiconservative replication.?

Section C, # 6. What amino acid sequence did your piece of DNA encode for?

Section D, # 4. What happened to the amino acid sequence when you changed one nucleotide base?