Course Code: BIO 024

LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

Activity Title: NUCLEIC ACIDS Materials:

Objectives: At the end of this activity, you should be able to Pen, Notes & LAS ( materials are
reflected in each activity)
1. Extract DNA from plant and animal cells.
2. Understand the structure of cells. References:
3. Identify the types of mutations, and explain the molecular effects  https://www.futurelearn.com/cour
that each has on base sequences of DNA ses/biochemistry
 http://www.dnaftb.org/
1.

The DNA molecule is shaped like a twisted ladder.

The DNA molecule is shaped like a twisted ladder.

Earlier work had shown that DNA is composed of building blocks called nucleotides consisting of a deoxyribose sugar, a
phosphate group, and one of four nitrogen bases — adenine (A), thymine (T), guanine (G), and cytosine (C). Phosphates
and sugars of adjacent nucleotides link to form a long polymer. Other key experiments showed that the ratios of A-to-T
and G-to-C are constant in all living things. X-ray crystallography provided the final clue that the DNA molecule is a
double helix, shaped like a twisted ladder.

In 1953, the race to determine how these pieces fit together in a three-dimensional structure
was won by James Watson and Francis Crick at the Cavendish Laboratory in Cambridge,
England. They showed that alternating deoxyribose and phosphate molecules form the
twisted uprights of the DNA ladder. The rungs of the ladder are formed by complementary
pairs of nitrogen bases — A always paired with T and G always paired with C.

RNA is an intermediary between DNA and protein.

DNA is found mostly in the cell nucleus, but another type of nucleic acid, RNA, is common in
the cytoplasm. Watson and Crick proposed that RNA must copy the DNA message in the
nucleus and carry it out to the cytoplasm, where proteins are synthesized. Crick also predicted
the existence of an "adaptor" molecule that reads the genetic code and selects the appropriate
amino acids to add to a growing polypeptide chain. This proposed flow of genetic information
from DNA to RNA to protein became known as the "Central Dogma."
As it turned out, several types of RNA are involved in the utilization of genetic information. In
the nucleus, the DNA code is "transcribed," or copied, into a messenger RNA (mRNA) molecule. In the cytoplasm, the
mRNA code is "translated" into amino acids. Translation is orchestrated at the ribosome — itself partly composed of
RNA — with transfer RNA playing the role of adaptor.

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

Activity # 1 DNA Extraction

Description: The students will use a banana fruit and their own cheek cells to extract
DNA.

Objectives:

1. To extract DNA from plant and animal cells.

2. To understand the structure of cells.

Length of Activity: 2 hours

A: DNA Extraction from Banana Fruit

Materials:

• 1 large banana cut into tiny pieces

• 1/2 cup distilled water
• A bowl of warm water
• 2 teaspoons clear, colorless (i.e., not cloudy) shampoo or liquid soap
• 1 teaspoon table salt
• 15 ml of 70% ethyl or isopropyl alcohol(i.e., rubbing alcohol) in 25 ml or 50 ml sealed bottle; chill the alcohol
by placing the test tube in a bowl containing ice cubes and some water 30minutes before the start of the
Experiment.
• zip lock or any clear plastic bag or a glass or any bowl
• tape (optional)
• 2 plastic spoons
• muslin cloth, thin cloth or a coffee filter
• clear glass
• medicine dropper (optional)
• toothpick or paper clip

Instructions:

1.Using a knife, cut the banana into tiny pieces to expose more of the cells.

2. Place the cut bananas in a glass with a spoon then smash the banana, but if you use a zip-top plastic bag. Add
the cut banana fruit to the bag remove the air, seal and then start squishing it up. Once it’s turned into a paste
set the bag aside.

3. Fill a clear glass with 1/2 cup of water. Slowly add in 2 teaspoons of dish soap and 1 teaspoon of table salt.
Gently mix this solution without making bubbles until the salt dissolves.

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

4. Add this solution to your zip lock bag of squished up fruit. Don’t add to much liquid. Add enough so that you
have a nice mixture that you cannot see through. That’s probably about 1/4 of a cup of soap solution. Flatten
out your baggie to remove most of the air and then seal it up. Gently squish the liquid around. If you have the
time, let this mixture sit for 10-20 minutes in bowl of warm water.

5. Place a coffee filter or a muslin cloth or thin cloth on top of a clear glass and carefully pour your fruit mixture
into the filter. You can gently squeeze the filter to get more liquid out, just don’t break it.

6. Take the ice cold alcohol and very slowly, drop by drop, pour it down the inside of the container with your
fruit mixture. You are trying to create a layer of alcohol that floats on the top of the water solution. The DNA
will come out of solution at the boundary layer between the water and alcohol. You should see some white
string almost cotton like strands begin to appear in the glass. That is the DNA! Use a hook (a bent paperclip
would work) or a tooth pick to slowly draw the DNA up and out of the solution.

7. Take a photo with yourself holding the bottle with banana DNA and post it in our designated platform.

Tips

When pouring the alcohol, make sure that two separate layers are being formed (The bottom layer being the
banana mixture and the top layer being the alcohol).

When extracting the DNA, twist the toothpick slowly. Be sure to only remove the DNA from the top layer.

B. Extraction of your Own DNA

Materials:

 Clear glass with cover ( or a jar with cover)

 Clear bottle with tight cover
 Lab Scoop or Spoon
 15 ml of 70% ethyl or isopropyl alcohol(i.e., rubbing alcohol) in 25 ml or
50 ml sealed bottle; chill the alcohol by placing the test tube in a bowl containing ice cubes and some
water 30minutes before the start of the experiment.
 Food coloring solution (any dark color if available only)
 Paper cup
 2 tsp Sodium chloride (salt)
 Distilled or bottled water
 1 tsp Liquid dish soap or hand soap
 Small glass vial(optional)

Instructions:

1. Make a saline solution in a clear glass by adding two teaspoons of salt to approximately 25 ml of distilled
water. Stir until the salt is completely dissolved.

2. Pour the saltwater into the paper cup.

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

3. Without swallowing, drink a mouthful of the solution from the paper cup and swish it back and forth for at
least 30 seconds, occasionally scraping your teeth along the inside of your cheeks as you do. It’s best to do this
with a clean mouth, i.e. not right after lunch.

4. Spit your mouthwash solution back into the cup. Then bend the cup into a sort of spout and pour the
mouthwashed solution into the test tube until it fills about one-half inch of the bottom of the test tube.

5. Carefully add two drops of the liquid soap.

6. Tilting the glass approximately 45 degrees Take the ice cold alcohol and very slowly, drop by drop, pour it
down the inside of the container without disturbing the solution. Since it’s less dense, the alcohol will sit atop
the mouthwash and soap solution. Then add 3 drops of food coloring (optional)

7. Tightly put the cap/cover of the jar and very slowly and gently tilt it upside down then right side up three
times. Do it carefully so as not to make bubbles.

8. Let the clear bottle or a jar sit undisturbed in an upright position for one minute. At this point, you should
begin to see a milky white thread, possibly interspersed with bubbles, appear between the solution and the
alcohol. That’s your DNA! After several minutes, the DNA should be suspended in the alcohol layer.

9. If you wish, insert a tooth pick or paper clip into the bottle or jar and gently wind the DNA around it.

10. Take a photo with yourself holding the bottle with your DNA and post it in our designated platform.

C. Using Plain Water instead of Saline Solution

Instruction: Follow all the steps in A excluding 1& 2 steps and swish only using Plain water.

Questions:

1. Why did you mash the banana fruit?

2. Why do we use liquid soap?

3. What does the salt do?

4. What does the cold ethanol do?

5. Why can’t you use room temperature ethanol?

6. How will you describe the appearance of the DNA you extracted both from banana fruit and your cheek cells.

7. Compare the results of A & B to C activity. Explain the result.

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

Mutations

Mutations are changes in genetic information.

The DNA sequences from two individuals of the same species are highly
similar — differing by only about one nucleotide in 1,000. Each DNA
difference results from a mutation — ranging from single nucleotide
changes, to small repeated units, to larger insertions and deletions. Some
mutations generate novel changes that are starting points of evolution,
and some are responsible for disease. In humans, the vast majority of
mutations occur in DNA regions that do not encode proteins. Most of
these are neutral in terms of evolution or health; they have no negative or
positive effect.
In the 1920s, DNA mutations were first induced in Drosophila using X-rays. Other types of ionizing radiation
were also found to produce mutations. Ultraviolet radiation, a component of sunlight, causes specific kinds of
DNA damage, including the linking of adjacent thymine nucleotides. Chemicals from a variety of man-made
and natural sources are known mutagens. Also, DNA replication, itself, is not perfect and is a source of new
mutations.

At the molecular level, mutations can be classified as being point mutations (affecting single genes) or
chromosomal mutations (changes in the arrangements of chromosomal DNA segments).
1. Point mutations are changes in single nucleotides and can be of 4 different types: (1) silent mutation (no
change in amino acid sequence specified by codons), (2) missense (change in amino acid sequence specified
by codons), (3) nonsense mutation (shortened amino acid sequence due to substitution causing a stop
codon to form somewhere in the mRNA), and (4) frame-shift mutation (addition/deletion of nucleotide
resulting in codon frame-shift).
2. Chromosomal mutations involve large regions of chromosomes and can be divided into 4 categories: (1)
deletion (loss of chromosomal segment), (2) duplication (deletion of segment from a chromosome and its
reattachment to its homologous chromosome), inversion (reinsertion of a broken chromosomal segment in
reverse order), and (4) translocation (exchange of chromosomal segments between non-homologous
chromosomes).

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

Activity # 2 Mutations

Objective: Identify the types of mutations, and explain the molecular effects that each has on base sequences
of DNA

Instructions:

A.Consider this original template DNA strand: 5’---AAGCAGCCATAACGAACGCAT---3’

1. For what sequence of amino acids does this DNA strand code? (assume it doesn’t contain introns)

2. The table below lists 5 different mutations that may occur in this DNA strand. What happens to the amino
acid sequence produced as a result of each mutation? (All positions are from the 5’ end of the DNA
template.)

Mutation Effect on amino acid sequence Type of mutation

Substitution of
T for G at
position 14

Insertion of T
between
positions 14
and 15

Deletion of C
at position 7

Substitution of
T for C at
position 4

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

B. Answer the following:

1. A template strand of a gene has this base sequence: 3´−TACAT*CCGATAGGGTCAT−5´. Exposure to

radiation causes a mutation in this gene. The thymine at the site of the mutation (*) is deleted. This will most
likely result in:

A. mRNA codons preceding (towards the 3’ end) the mutation to be misread

B. mRNA codons downstream to the start codon to be misread
C. no change of any kind
D. no change in the polypeptide coded by this gene
E. the AUG triplet functioning as a chain terminator

2.Which of the following is analogous to a frameshift mutation?

A. THERATATETHECAT
B. THETACATETHERAT
C. THECATARETHERAT
D. THECATATTHERAT
E. CATATETHERAT

3. Assume that the following polypeptide chain is produced by a wild-type gene in an organism:

N-met-lys-trp-leu-his-ala-glu-gly-lys-C

Assume that a phenotypically observable mutation occurred in the coding region of the mRNA
th
that coded for this polypeptide. Assume that a base substitution occurred in the 7 base position
counting from the 5' end of that coding region. Without referring to actual codons, what is a
likely sequence resulting from such an alternation?

A. N-met-lys-arg-leu-his-ala-glu-gly-lys-C
B. N-met-C
C. N-met-lys-trp-leu-his-ala-ala-gly-lys-C
D. N-met-lys-trp-leu-his-ala-glu-gly-lys-C
E. None of the above

SWU PHINMA, College of Pharmacy 2020

 Course Code: BIO 024
LAS # 6
BIOCHEMISTRY LABORATORY
NUCLEIC ACIDS

4.

5.

SWU PHINMA, College of Pharmacy 2020