[VOL1, ISSUE 1] 2014

Journal of Biochemistry

Experiment Article

DNA ISOLATION FROM MUSA ACUMINATA (AA Group)

USING RAPID SIMPLE METHOD OF ISOLATION
1

Ayang, Noel Angelo , Garlan, Paul Trishan , Jerusalem, Rod Florence , Sy, Elaine

BS Chemistry 3-2, Department of Physical Sciences, College of Science, Polytechnic University of the Philippines,
Sta. Mesa, Manila

ABSTRACT:
Just like us, banana plants have genes and DNA in their cells, and just like us, their DNA determines
their traits. The soft flesh of a banana provides a ready source of DNA. Using a few simple purification steps
in a classroom setting, students can yield loads of crudely prepared DNA. The experiment presents the
isolation of DNA from banana. First, banana was crushed and detergent /salt solution was added for the cell
membrane to be destroyed and for the nuclear membrane to be released. Then, it was reconstituted,
precipitated by alcohol and chemically disrupted. Finally it was put to UV-Vis for the DNA to be determined.
The result does not meet the accepted A260/A280 ratio for the DNA purity, which is 1.7-2.0, for it was below
1.5. DNA is required for many applications. Proper laboratory practice and care must be taken to ensure
reliable and reproducible results.

Keywords: DNA, Isolation, Extraction, Nucleic Acid, Reconstituion

INTRODUCTION
What do you have in common with a
banana? Even though we might not look
alike, all living thingsbananas and people
includedare made up of the same basic
material.

blocks called cells. Within an organism,

each cell contains a complete set of
"blueprints". These directions determine the
organism's characteristics.

Just like houses are made up of smaller

units such as bricks, all living things are
made up trillions of microscopic building
Polytechnic University of the Philippines
S.Y. 2014-2015
Page 1

[VOL1, ISSUE 1] 2014

Deoxyribonucleic
acid
(DNA)
is
a molecule that
encodes
the genetic
instructions used in the development and
functioning
of
all
known
living organisms and many viruses. It is one
of the most important parts of the cell
because it contains the instructions for the
synthesis of proteins and RNA inside the
cell. With its absence, cell cannot function.
Most DNA is located in the cell nucleus
(where it is called nuclear DNA), but a small
amount of DNA can also be found in the
mitochondria
(where
it
is
called mitochondrial DNA or mtDNA). A
DNA sequence is a specific lineup of
chemical base pairs along its strand. The
part of DNA that determines what protein to

produce and when, is called a gene. Genes

control everything from hair color to blood
sugar by telling cells which proteins to
make, how much, when, and where.
DNA isolation is a process of purification of
DNA from sample using a combination of
physical and chemical methods. Purified
DNA has many applications in our modern
Polytechnic University of the Philippines
S.Y. 2014-2015
Page 2

[VOL1, ISSUE 1] 2014

world. It could be used for Genetic
engineering in which they modify DNA
sequence for practical application such as in
agriculture and in medical research. It could
also be used in forensics which forensic
scientists
identify
bodies,
crosscontamination of evidence. And it has many
other applications.

the DNA and separated it from the sample

through decantation. The separated DNA is
then washed with water and tested its purity
through
the
use
of
UV-Vis
spectrophotometer.
General Objective: The experiment aims to
extract completely the DNA from the
banana.

In this paper, we extracted DNA in the fruit

of Musa acuminata (AA Group) using a
rapid method of isolation by disrupting the
cell wall with the use of blender, and
separating the DNA from its contaminants
by the use of alcohol. We have precipitated

Specific Objective: The experiment aims to

extract completely the DNA from the banana
using a rapid simple method of isolation.
Fig 1: Mechanical
Membrane

Disruption

of

Cell

METHODOLOGY
The laboratory equipments used in the
experiment were test tubes, stirring rod, 250
mL beakers, 500 mL beaker, pipette,

SF

ACMIt RHi
nmalaone
Drslim
ptcsc
tdoho
ropCg
uhMones
mbCon
eMti
nlbiz
tPea
arut
teit
icoi
on
nio
pn
i

h
n
t
f
r

i
et
f
r

e
i

s
s

o
i

a
e

aspirator, watch glass and inoculating loop.

Additional materials are blender, salt and
dishwashingliquid.

l
l

Polytechnic University of the Philippines

Page 3

t
i
S.Y. 2014-2015
o
n

o
m

c a

l
m

u
u

n
e

a l

[VOL1, ISSUE 1] 2014

Fig 2. Schematic diagram of the procedure

Mechanical Disruption of Cell Membrane

Chemical Disruption of Cell Membrane

Using your knife, cut your banana into tiny

pieces to expose more of the cells. Place
your banana pieces in the blender. Add
some ice and mix in the blender for some
seconds.

Add a teaspoon of salt. Mix it again. The

salt will help the DNA stay together during
the mashing process. After mixing, add
small drops of dishwashing liquid and gently
stir the mixture. You should try not to create
bubbles when stirring. The soap helps to
break-down cell membranes to release the
DNA.

Homogenization
Add distilled water. Mix in the blender for 5
to 10 seconds making sure the mixture is
not too runny.

Alcohol Precipitation
Transfer an aliquot of the mixture using
pipette into test tubes, about of its size.
Carefully pour very cold 95% ethanol down
the side of the glass stopping near the top.
Let it sit undisturbed for about four to five
minutes or so in a 500mL beaker with ice.
Do not shake. The white material coming
out of solution as a precipitate is DNA.

Reconstitution
Dip the glass rod into the tube, slowly
rotating it to spool out the bananas DNA.
Using the inoculating loop, scoop out the
DNA precipitate. Put the precipitate in a
watch glass and allow the remaining alcohol
to evaporate. After a minute, reconstitute it
on water to remove excess alcohol that is
not
completely
eliminated
through
evaporation.

Fig 3: Sample mixture in test tubes

Polytechnic University of the Philippines
S.Y. 2014-2015
Page 4

[VOL1, ISSUE 1] 2014

Instrumentation
After
reconstitution,
we now test the
DNAs
purity

through the use of UV-Vis Spectrometer.

Make a 3mL sample mixture of DNA and
water into a new test tube. Setting the
wavelengths to 260nm and 280nm, we now
read and record its absorbance.

Fig 4: Adding of dishwashing liquid

Result and Discussion

Nucleic Acid
absorbs
ultraviolet light in
a specific pattern. The collected DNA
samples undergo a spectrophotometric
analysis. Table 1 shows the Absorbance of
the samples @ 260 nm and 280 nm.

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 5

[VOL1, ISSUE 1] 2014

Sample

Absorbance
nm

260 Absorbance @ 280 nm

0.422

0.378

0.266

0.221

0.008

0.006

Table 1. Absorbance of collected DNA samples from Banana @ 260 and 280 nm.
DNA concentration estimation by Spectrophotometric Absorption
Quantitation of Nucleic Acid were obtained
using the formula:

Concentration (g/mL) = Abs@260 50

g/mL dilution factor

At a 1-cm path length, the Absorbance

(optical density) at 260 nm (OD260) equals
1.0 for a 50 g/mL solution of double
stranded DNA. Table 2 shows the calculated
concentration of the isolated pure DNA
samples from Banana.

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 6

[VOL1, ISSUE 1] 2014

Sample

Concentration (ppm)

42.2 ppm

37.8 ppm

0.8 ppm

Table 2.Concentration of collected DNA samples from Banana @ 260 nm.

Nucleic acids and proteins have

absorbance maxima at 260 and 280 nm,
respectively. The ratio of absorbance at
these wavelengths has been used as a
measure of purity in both nucleic acid and
protein extractions. A ratio of ~1.8 is

generally accepted as pure for DNA; a

ratio of ~2.0 is generally accepted as pure
for RNA. Table 3 shows the calculated ratio
of absorbance at 260/280 nm from the
isolated DNA samples.

Sample

Absorbanc
e @ 260 nm

Absorbanc
e @ 280 nm

Ratio
(Abs
260/280)

0.422

0.378

1.1

0.266

0.221

1.2

0.008

0.006

1.3

Table 3.The ratio of absorbance obtained at wavelength 260 and 280 nm.

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 7

[VOL1, ISSUE 1] 2014

Discussion

Extraction of DNA from Banana

concentration of the double stranded DNAs

present in the sample. Table 1.2 shows the
calculated concentration of isolated DNA
sample
using
the
formula:
Concentration (g/mL) = Abs@260 50
g/mL dilution factor

Banana was use as source of DNA since

bananas are soft and dense, without a lot of
stringy or gritty material which might be
present in some fruits
(a pear, for instance). Their softness makes
it easy to release their DNA without a lot of
work. Crushing of bananas by the use of
blender helps break down the tough walls of
the cells to release the cell contents.
Addition of detergent to the slurry helps to
break down the cell and nuclear membrane.
Detergent is made up of Sodium Laurel
Sulfate, which dissolve the fats and lipid
bilayer of membranes and release the
cellular contents, including DNA. Once the
DNA is released from the cells, the addition
of salt to the solution enables the DNA
strands to come together, or aggregate.
Since DNA is not soluble in alcohol, addition
of cold Ethanol dehydrates the DNA by
removing the water. This dehydrated
molecule then forms the DNA precipitate,
while the other remaining materials remain
in the solution.

Assessment of Nucleic Acid purity

The
ratio
of
absorbance
at
wavelength260 and 280 has been used as a
measure of purity in both nucleic acid and
protein extractions. It is important to note
that the A260/A280 ratio is only an
indication of purity rather than a precise
answer. A ratio of ~1.8 is generally accepted
as pure for DNA; a ratio of ~2.0 is
generally accepted as pure for RNA.

A260/280 ratio present at Table 3 do not

met the generally accepted ratio for a pure
DNA. The values of the ratio that are
obtained are relatively smaller than of the
standard ratio for a pure DNA.
Fig 5: Extracted DNA

Concentration of isolated DNA samples

The calculated concentration of DNA

samples from Banana accounts only for the

Several factors influence the accuracy of

our A260/A280 ratio.A low A260/A280 ratio
may be caused by residual phenol or other
reagent associated with the extraction, and

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 8

[VOL1, ISSUE 1] 2014

a very low concentration (> 10 ng/ul) of

nucleic acid. In the case of our experiment,
the extraction of DNA from banana was
properly done but in some cases, it may be
contaminated by other reagents or
substances. During the spooling of DNA
precipitates from the test tube, some of the
bananas cell components were also spool
out of the test tube together with the DNA
precipitates which causes the inaccuracy of
our A260/280 ratio. Moreover, the DNA
concentration of the samples may also
influence the inaccuracy of our result. Due
to a very small amount of DNA samples that
were collected, it reflected to the very low
concentration of DNA present in our
samples.

Wavelength
Accuracy
Spectrophotometers

of

the

Although the absorbance of a nucleic acid

at 260 nm is generally on a plateau, the
absorbance curve at 280 nm is quite steeply
sloped. A slight shift in wavelength accuracy
will have a large effect on 260/280 ratios. It
is possible to see as much as a 0.4
difference in the 260/280 ratio when
measuring the same nucleic acid sample on
two spectrophotometers that are both within
a 1 nm wavelength accuracy specification.
Two different types of Spectrophotometers
may measure two different readings.
Nucleotide Mix in the samples
The type(s) of protein present will also
have an effect. Absorbance in the UV range
by proteins is primarily the result of aromatic
ring structures. Proteins are composed of
22 different amino acids of which only three
contain aromatic side chains. Thus the
amino acid sequence of proteins would be
expected to influence the ability of a protein
to absorb light at 280 nm. For example
bovine serum albumin (BSA) has an
extinction coefficient value of 0.7 for a 1
mg/ml solution at 280nm, while streptavidin
has an extinction coefficient of 3.4,
absorbing almost five times as much light at
280nm at the same concentration.
Due to the different absorption spectra,
the nucleotide composition of the bases
present in DNA will have different
A260/A280 ratios.

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 9

[VOL1, ISSUE 1] 2014

Nucleotide

A260/A280 ratio

Adenine

4.50

Cytosine

1.51

Guanine

1.15

Thymine

1.47
Table 4.A260/A280 ratios for nucleotides.

Therefore the ratio will be approximately

equal to the weighted average of the
A260/A280 ratios estimated for each
nucleotide if measured independently, which

Conclusion

Isolation/extraction of high quality, intact

pure DNA is required for many applications.
Proper laboratory practice and care must
be taken
and reproducible
Fig to
6: ensure
Alcohol reliable
precipitation
results. A good quality DNA sample should
have a A260/A280 ratio of 1.7-2.0 and an

explains why the accepted ratio of 1.8 for

pure DNA is an approximation. The actual
ratio will depend on the composition of the
nucleic acid.
A260/A230ratio of greater than 1.5, but since
the sensitivity of different techniques to
these contaminants varies, these values
should only be taken as a guide to the purity
of your sample. Our result do not met the
general accepted ratio for a pure DNA.
The reasons for this inaccuracy may cause
by an error in the preparation and extraction
of the DNA precipitates and the presence
ofother reagents or contaminants thatcause
a low A260/280 ratio of our DNA samples.To
improve the accuracy of DNA concentration
determination, allowance should be made
for any impurities in the solution. This can
be estimated by adjusting the A260
measurement for turbidity which is
measured at an absorbance of A320. A
reading at 320nm will indicate if there is
turbidity in the solution, another indication of
possible contamination. Therefore, taking a
spectrum of readings from 230nm to 320nm
is one of the suggested and informative
ways to accurately determine the purity and
concentration of DNA.

REFERENCES
Polytechnic University of the Philippines
S.Y. 2014-2015
Page 10

[VOL1, ISSUE 1] 2014

1. http://www.sciencedaily.com/articles/
d/dna.htm
2. http://ghr.nlm.nih.gov/handbook/basi
cs/dna
3. Dahm, R (January 2008).
"Discovering DNA: Friedrich
Miescher and the early years of
nucleic acid research.". Human
Genetics 122 (6): 565
81. doi:10.1007/s00439-007-0433-0.
PMID 17901982.

Polytechnic University of the Philippines

S.Y. 2014-2015
Page 11