Vol1, Issue 1
[Vol1, Issue 1]December 2014
Journal of BiochemistryPolytechnic University of the
PhilippinesS.Y. 2014-2015Page 1
Experiment ArticleDNA ISOLATION FROM MUSA ACUMINATA (AA Group)
USING RAPID SIMPLE METHOD OF ISOLATIONAyang, Noel Angelo1, Garlan,
Paul Trishan2, Jerusalem, Rod Florence3, Sy, Elaine4BS 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. Thesoft
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, ReconstituionINTRODUCTION
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.Just like houses are made up of
smaller units such as bricks, all living things are made up
trillions of microscopic building blocks called cells. Within an
organism, each cell contains a complete set of "blueprints". These
directions determine the organism's
characteristics.Deoxyribonucleic acid (DNA) is amoleculethat
encodes thegenetic instructions used in the development and
functioning of all known livingorganismsand manyviruses.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
calledmitochondrial DNAor 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
isolationis a process of purification of DNA from sample using a
combination of physical and chemical methods. Purified DNA has many
applications in our modern 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, cross-contamination of evidence. And it has many other
applications.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 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.Specific Objective: The experiment aims to extract
completely the DNA from the banana using a rapid simple method of
isolation.Fig 1: Mechanical Disruption of Cell Membrane
METHODOLOGY
The laboratory equipments used in the experiment were test
tubes, stirring rod, 250 mL beakers, 500 mL beaker, pipette,
aspirator, watch glass and inoculating loop. Additional materials
are blender, salt and dishwashingliquid.
Fig 2. Schematic diagram of the procedure
Mechanical Disruption of Cell MembraneUsing 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.HomogenizationAdd distilled water. Mix in the
blender for 5 to 10 seconds making sure the mixture is not too
runny.Chemical Disruption of Cell MembraneAdd 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.
Alcohol PrecipitationTransfer 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. ReconstitutionDip 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 tubesInstrumentationAfter
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.
SampleAbsorbance @ 260 nmAbsorbance @ 280 nm
10.4220.378
20.2660.221
30.0080.006
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.
SampleConcentration (ppm)
142.2 ppm
237.8 ppm
30.8 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.
SampleAbsorbance @ 260 nmAbsorbance @ 280 nmRatio (Abs
260/280)
10.4220.3781.1
20.2660.2211.2
30.0080.0061.3
Table 3.The ratio of absorbance obtained at wavelength 260 and
280 nm.
Discussion
Extraction of DNA from Banana
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.
Concentration of isolated DNA samples
The calculated concentration of DNA samples from Banana accounts
only for the 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
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
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 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 of the Spectrophotometers
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.
NucleotideA260/A280 ratio
Adenine4.50
Cytosine1.51
Guanine1.15
Thymine1.47
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 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.
Conclusion
Fig 6: Alcohol precipitationIsolation/extraction of high
quality, intact pure DNA is required for many applications. Proper
laboratory practice and care must be taken to ensure reliable and
reproducible results.A good quality DNA sample should have a
A260/A280ratio of 1.7-2.0 and an 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
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2. http://ghr.nlm.nih.gov/handbook/basics/dna
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and the early years of nucleic acid research.".Human
Genetics122(6):
56581.doi:10.1007/s00439-007-0433-0.PMID17901982.