DISCUSSION

For an overall background, it can be justified that DNA is actually the genetic
information in organisms that codes for proteins and controls the cells function. The DNA is
a double helix molecule that is formed by various nucleotides ( Adenine, Thymine, Guanine,
Cytosine) with a sugar phosphate backbone. Different organisms can have larger or smaller
genomes. DNA is also what makes up chromosomes during cell replication.
DNA extraction refers to the process that scientists use to break down a piece of
evidence, such as a piece of hair or a drop of blood, in order to determine the individuals
DNA, which is unique to each person.
In this experiment, Kiwi fruit is used to isolate DNA from the cells as kiwi fruit has a
very much common in human. It was said a kiwis genetic material is very similar to human.
QUESTIONS :
1. What was the purpose of the detergent?
Each cell is surrounded by a sack ( the cell membrane). DNA is found inside the
second sack (nucleus) within each cell. To see the DNA we have to break it open. A cell
membrane has 2 layers of lipid (fat) molecules with protein going through them. When the
lysis buffer ( detergent) comes close to the cell, it captures the lipids and the proteins which
is in other words breaking open the cell destroying the fatty membrane. As a result, DNA is
now released into the solution.

2. What was the purpose of the salt?
The usage of the salt is also necessary as it helps to remove proteins that are bound to the
DNA . It also helps to keep the proteins dissolved in the aqueous
layer so they dont precipitate in the alcohol along with the DNA.





3. Why was it necessary to mash the fruit?
While conducting the experiment, the fruit is mashed very well. It is because we need
to get the DNA out from the nucleus and into the liquid that will drip into the test tube.

4. Why do you heat and cool the mixture?
When we heat the mixture, it causes the cell walls to break down and exposing the cell
membranes, thus making is easier for us to extract the DNA present. Once the nuclear
membrane is destroyed by the soap, the DNA is now exposed to a restriction enzyme that
destroy DNA present in the cells cytoplasm, and the DNA will may quickly be degraded.
However, cooling the solution slows down the process of degradation because the enzyme is
temperature sensitive.

5. What happen when cold alcohol is added?
Alcohol is less dense than water, so it floats on top. DNA is not soluble in alcohol
while the other cell parts are. By adding alcohol, DNA precipitates out of the solution and
collect at the interface of the alcohol and washing up liquid. The colder the alcohol the less
soluble the DNA will be in it.

As a matter of fact, DNA dissolves in water but precipitates in alcohol. Cold
alcohol is used to separate DNA out of water-based solutions. This allows the DNA to be
purified for subsequent genetic testing. Adding alcohol to a solution containing DNA is a
simple way to obtain the pure DNA required, and colder temperatures slow down enzymes
that can break down DNA, giving better extraction results.

6. Would it make a difference if warm alcohol is added?
If warm alcohol is added, it will surely makes a difference as warm alcohol will
increase the solubility of the DNA in it, making it hard to extract the DNA as it is difficult to
clump together.

From the experiment, we can observe the DNA as a long, stringy molecule. This
happens as the salt that we added helps it to stick together. The thing that we see is actually
the clumps of tangled DNA molecules. DNA normally stays dissolved in water, but when
salty DNA comes into contact with alcohol, it becomes undissolved. This is what we called
as PRECIPITATION. The physical force of the DNA clumping together as it precipitates
pulls more strands along with it as it rises into the alcohol as what I have mentioned earlier.

Based on the experiment too, it is crystal clear that the DNA extraction is
definitely an important biology procedure. With the ability to remove DNA from an
organism, scientists can observe, manipulate, and classify the DNA. Scientists can
identify genetic disorders or diseases from studying DNA. Scientists can possibly find cures
for these causes by manipulating or experimenting with this DNA. Scientists can accurately
sort organisms into classes because of DNA uniqueness. If we didn't have DNA extraction,
it would be a lot harder to decide which organisms are different from each other. Scientists
can genetically engineer some organisms to produce beneficial things. A common example
is that of insulin. Scientists can genetically engineer insulin production so that people with
diabetes can live longer.
















6.0 DISCUSSION
During interphase, the chromosomes are not visible. During prophase, the
chromosomes appear because it is condensed. During metaphase, the chromosomes
appear to line up along the equator of the cell. During anaphase, most of the
chromosomes are seen at the opposite pole of the cell with some of them still moving
towards it. During telophase, all chromatids are seen to have reached opposite poles and
the nuclear membrane reforms.
We were supposed to observe the following image :
Prophase

Metaphase

Anaphase

Telophase


However, we only managed to observe as shown in result, which may be caused by
factors such as :
a) Too much staining was used, causing the specimen to seem very dark and hard to
identify any diving cell
b) Over-heating of the specimen and caused the specimen to extremely dried out
c) The root tip was not squeezed enough when we press it with thumb and so the
organelles was not spread
d) The root tips was not handled gently and the great force has caused the root tip
break and destroy the structure of the cell

Blue toluidine is used to stain the chromosomes by penetrating into the nucleus.
Blue toluidine is a basic thiazine metachromatic dye with high affinity for acidic
tissue components, thereby staining tissues rich in DNA and RNA. It has found wide
applications both as vital staining in living tissues and as a special stain owing to its
metachromatic property.
The onion root is crushed into small pieces so that no large pieces are observable
on the slide. It is also to enable us to capture many cells in different phases of the cell
cycle, which are prophase, metaphase, anaphase as well as telophase, as the cycle is a
continuous process.
The purpose of pressing the specimen with a piece of paper is to obtain a single
layer of cells and dissolve the middle lamella of the cell completely. This steps is
important to avoid the cell from overlapping at some part in the microscope slide.





a) Monohybrid cross
Phenotype Genotype O E (O-E) (O-E)
2
(O-E)
2
/E
Tall TT 33 30 3 9 0.3
Short Tt 7 10 -3 9 0.9
X
2
=1.2

X
2
1.2
Degree of Freedom 1
Critical Value 1.07
Probability 0.3
Range 0.20 < X
2
< 0.30

Hypothesis :
Since P value (0.3) is larger than 0.05, the hypothesis F2 ratio (3:1) for monohybrid cross
is accepted.














b) Dihybrid cross
Phenotype Genotype O E (O-E) (O-E)
2
(O-E)
2
/E
Tall, purple
flower
TTAA/
TtAa
29 22.5 6.5 42.25 1.878
Tall, white
flower
Ttaa/TTaa 5 7.5 -2.5 6.25 0.833
Short,
purple
flower
ttAA/ttAa 4 7.5 -3.5 12.25 1.633
Short,
white
flower
ttaa 3 2.5 0.5 0.25 0.1
X
2
=4.444

X
2
4.444
Degree of Freedom 3
Critical Value 3.66
Probability 0.3
Range 0.20 < X
2
< 0.30

Hypothesis :
Since P value (0.3) is larger than 0.05, the hypothesis F2 ratio (9:3:3:1) for dihybrid cross
is accepted.











In this experiment, we used different colored cards to represent different chromosome.
For monohybrid crossing which only involves one trait, the cards used are:
Color of card Gamete represent Trait
Blue T Tall
Yellow t Short

While for dihybrid crossing,
Color of card Gamete represent Trait
Blue T Tall
Yellow t Short
Green A Purple flower
Red a White flower

The cards are mixed in a beaker and a pair of each trait is chosen randomly to represent
the random combination during cell division.
Based on our experiment, the phenotype ratios are:
For monohybrid cross,
Characteristics Number of pairs
Tall 33
Short 7

For dihybrid cross,
Characteristics Number of pairs
Tall, purple flower 28
Short, purple flower 4
Tall, white flower 5
Short, white flower 3








The chi-squared value can be obtained from the chi-squared table.
A higher accuracy is when the observed value, (O) is equal to the expected theoretical
value (E), which makes the value of X
2
to be 0.
We can determine whether the deviation is within the expected limit based on the limit
for occurrence by chance set by statisticians, which is 1 in 20 = 0.05. The value becomes
the critical value which means, if the probability obtained is within the range, our
experiment is said to be successful.
In our experiment, we managed to obtain 0.3 for monohybrid and dihybrid crosses which
makes our experiment has succeed to prove that the probability for Mendelian F2 ratio is
3:1 and 9:3:3:1 respectively.












To collect a set of karyotype, we can obtain a sample which will initiate mitosis, such as
blood. Then, chromosome from the sample will arrested in metaphase state by addition of
colchicines which will also make them short and fat thus it is clearly arranged form.
The image of the chromosome is then taken by using the combination of computer and
light microscope. When we print out the image, this is what the scattered chromosomes
from the handouts represents.
The chromosomes on results shown that it is arranged from the biggest to the smallest,
based on criteria such as location of centromere, presence of satellite, and the type of the
short arm.
To differentiate male and female, we can find the X and Y chromosomes on the 23
chromosome ; if it consists of XX, the karyotype belongs to female, which if it consists of
XY, the karytope belongs to male.
For the six sets provided, Set A, B, C, and D has a disease that can be determined by
observing the abnormalities on any of the chromosomes. For another two sets, they are
one normal male karyotype and one normal female karyotype.