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The experiment aims to isolate DNA from microbial, plant and animal sources, it also aims to determine
the purity of isolated DNA and characterize DNA following acid hydrolysis. DNA was isolated from the
plant source, onion. Absorbance was measure under the wavelengths 260nm and 280nm which was
followed by hydrolysis using hydrochloric acid after which was subjected to different qualitative color
reaction: Dishe Test, a test for deoxyribose, Test for Phosphates, Murexide test, a test for Purines and
Wheeler-Johnson Test, a test for Pyrimidines. The computed absorbance ratio is 0.56. Dische Test
produced a blue solution, Test for Phosphates produced a yellow precipitate, Murexide test yields a
yellow to red residue while Wheeler-Johnson test yields a pale yellow turbid solution.
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twisted about each other in a double helix. Both
chains are right handed however, since each
Nucleic acids are the biological strand has both free 5͛ hydroxyl group at one
molecule essential for life. They make up the end and a free 3͛ hydroxyl on the other end,
most important macromolecules. They are high each strand has a polarity or directionality. The
molecular weight biopolymers of polarity of the two strands of the molecule is in
mononucleotides. The backbone of a opposite directions and thus, DNA is described
nucleic acid is made of alternating sugar and as anti-parallel structure.
phosphate molecules bonded together in a long
chain. Each of the sugar groups in the backbone
is attached to a third type of molecule called
a nucleotide base. Though only four
different nucleotide bases can occur in a nucleic
acid, each nucleic acid contains millions of bases
bonded to it. The order in which these
nucleotide bases appear in the nucleic acid is
the coding for the information carried in
the molecule.
ÑV h $
!'() The next step is followed by
h " deproteinization. Deproteinization involves
A 0.5 ml of nucleic acid solution was treated adding a protease enzyme Papain, a common
with an excess of bromine water until the enzyme used to clean soft contact lenses. This
will denature the proteins clinging to the DNA Table2. Results of Qualitative Color Reaction of
making the molecule flexible and easy to spool. Acid hydrolyzed DNA
Precipitation of DNA involves adding ethanol h h
alcohol which causes every component in the Dische Test Light Blue Blue solution
filtrate to stay in solution except DNA. solution
Test for Formation of Formation of
Table1. DNA isolation Phosphates Yellow yellow
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precipitate precipitate
% Murexide Formation of Formation of
Plant DNA Light Yellow Test red precipitate yellow to red
turbid Solution precipitate
Wheeler- Formation of Pale yellow
V 4 c johnson Test Purple turbid
precipitate solution
The measurement of the absorbances
allows measurement of the DNA *V h
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concentration and provides information
about the contaminant levels. DNA absorbs DNA can be identified chemically with the
light most strongly at 260nm so the Dische diphenylamine test. The reaction
absorbance value at this wavelength (called between the Dische reagent and 2-
A260) can be used to estimate the DNA deoxypentose results in the development of
concentration while the absorbance at a blue color. The reaction depends on the
280nm is used as an indicator of protein conversion of the pentose to w-
contamination. A good quality DNA sample hydroxylaevulinic aldehyde which then
should have an A260/A280 ratio of 1.7-2.0 reacts with diphenylamine to give a a blue
colored complex (test tube 1 and 2). The
intensity of the blue color is proportional to
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the concentration of DNA.
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DNA is generally quite stable. It will resist
attack in acid and alkali solutions. However,
in mild acid solutions - at pH 4 - the beta-
glycosidic bonds to the purine bases are
hydrolyzed. Protonation of purine bases
occurs at this pH. Protonated purines are
good leaving groups hence the hydrolysis.
Once this happens, the depurinated sugar
can easily isomerize into the open-chain Figure3. Dische Test Reaction
form and in this form the depurinated (or
apurinic) DNA is susceptible to cleavage by #V h $ %
hydroxyl ions.
In the test for presence of phosphates for
DNA, a yellow precipitate was obtained. The
ammonium molybdate solution reacted
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with the sample which yields yellow
crystals, phosphoammonium molybdate
which is a positive result. +. -
7
Anthony Carpi. Nucleic Acids: DNA and RNA.
&V h $
!'() Retrieved February 9, 2011 from:
h " http://www.visionlearning.com/library/module
Bromine water reacted with the sample to _viewer.php?mid=63
form 5-bromo-6hydroxyhydroxo derivative
8
which produces a green coloration. Upon Bagsican, MJ. DNA isolation from onion,
addition of Ba(OH)2 will give a result of Ultraviolet Measurement of Isolated DNA and
purple precipitate. Chemical Characterization of DNA. Retrieved
V February 9, 2011 from:
ÑV h $ ! h " http://www.scribd.com/doc/29380667/DNA-
Isolation-From-Onion-Ultraviolet-
In the test for presence of purines, DNA is Measurement-of-Isolated-DNA-and-Chemical-
reacted with Nitric acid since Purines are Characterization-of-DNA
known to be readily soluble in dilute acid.
9
Nitric acid oxidized it leaving a yellow Keush, P. Test for Deoxyribose in DNA.
precipitate upon evaporation; however it Retrieved February 10, 2011 from:
turned red when moistened with a base, a http://www.uni-
positive result for presence of purine bases. regensburg.de/Fakultaeten/nat_Fak_IV/Organis
che_Chemie/Didaktik/Keusch/p31_d_rib-e.htm
10
+, - Klibaner, E. Extraction of DNA from White
1
Abraham, C. (1962). Biochemistry 3rd Ed. Onion. Retrieved February 10, 2011 from:
Saunders Company: Philadelphia London pp.99- http://www.scienceteacherprogram.org/biolog
114 y/oniondna.html
2
Boyer, R. (2006). Concepts in Biochemistry 3rd 11
Kuddi, S. DNA isolation, Restriction,
edition. Hoboken NJ: John Wiley & Sons Inc. Visualization and Quantification. Retrieved
pp.282-311 February 9, 2011 from:
http://www.scribd.com/doc/14614699/DNA-
3
Crisostomo, AC., Daya, ML., de Guia, RM, et al. isolation-restriction-visualitation-and-
(2010). Laboratory Manual in General quantification
Biochemistry. Quezon City: C&E Publishing Inc.
12
pp 55-63. Nucleic Acid. Retrieved February 9, 2011 from:
http://en.wikipedia.org/wiki/Nucleic_acid
4
Hecht, S. (1996). Biorganic Chemistry Nucleic
13
Acid. Oxford University Press: New York pp.29- The physical and chemical properties of
35 nucleic acids. Retrieved February 10, 2011 from:
http://www.mun.ca/biochem/courses/3107/To
5
Jack, R. (1995). Basic Biochemical Laboratory pics/DNA_properties.html
Procedure and Computing. Oxford University
Press: New York.pp100-103
6
Wilson, K. (2010). Priniciples and technique in
Biochem and Molecular Biology 7th
Ed.Cambridge University Press: New York.
Pp139-176,187-194,195-198