ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS

Manalastas,J.M ,Manganaan,V.P, Mangilit,A.A, Medina,G.P, Merina.,A.N,

Department of Pharmacy, Faculty of Pharmacy

University of Santo Tomas

ABSTRACT

Gluten is a protein composite that appears in foods processed from wheat, barley,
and rye. Gluten helps foods maintain their shape, acting as a glue that holds food together.The
intact protein was isolated by washing the dough with water which removes starch. Gluten was
subjected to the different color reaction tests namely, Biuret, Ninhydrin, Xanthoproteic, Millon,
Hopkins-Cole, Sakaguchi, Nitroprusside, Pauly, Fohls and Amide test which determined the
structure, functional group and presence of amide and aromatic side chains. The intact protein
was also alkaline hydrolyzed, and the hydrolyzed protein was also subjected to the different
color reaction tests.

INTRODUCTION

Nucleic acids are biomolecules that allow
organisms to transfer genetic information
from one generation to the next. There are
two types of nucleic acids: deoxyribonucleic
acid (better known as DNA) and ribonucleic
acid(better known as RNA).
The backbone of a nucleic acid is made of
alternating sugar and phosphate.
In most living organisms (except for
viruses), genetic information is stored in
the molecule deoxyribonucleic acid. DNA is
a double stranded nucleic acid. It is
compose of nitrogenous bases such as
guanine, adenine, thymine, and cytosine.
The Nucleotide sequence has the
information found in the DNA and this
nucleotide is has a unique complimentary
nucleotide, each strand contains all the
information required to synthesize a new
DNA molecule.

The Four different nucleotide bases occur in

DNA: adenine (A), cytosine (C), guanine
(G), and thymine (T). Guanine and adinine
are have purine as their nitrogenous bases
while for cytosine and thymine its
pyrimidines.

In forming the DNA there are base pairs

Figure 1. DNA structure There are two important reasons why DNA
is composed of complementary bases. The
first is that only with complementary bases
can the two strands of DNA have
complementary sequences. Without the
complementary sequences DNA replication
as we know it would be impossible. The
second reason is that the pairs of bases are
complementary because their chemical
structure allows for strong hydrogen
bonding and they fit together well.
METHODOLOGY
A. Materials
The material needed in isolating the
DNA from onion was two(2) yellow onions. A
homogenizing solution compose of 5%
sodium dodecyl sulphate or sodium lauryl
sulphate (SDS), 0.15 M NaCl (sodium
chloride), 0.15 M sodium citate and 0.001 M
EDTA. For acid hydrolysis 1M HCL, 1M
KOH and glacial acetic acid was the
reagents. The glacial acetic acid was used
in adjusting the PH value of thr solution.
For the deoxyribose test or also known as
the Dische test, along with the DNA sample
diphenylanamine reagent was used.
In the test for purines concentrated HNO3
and 10% of potassium hydroxide was used
and guanine and adenine are tested . While
in pyrimidines where cytosine and uracil
was tested, it is treated by the bromine
water and barium hydroxide was the
reagent used.
The UV Vis/ Microplate was used for the
ultraviolet measurement of the isolated DNA
B. Procedure
1. DNA isolation from onion
In a 125-ml Erlenmeyer flask 50 ml of
the homogenizing solution was added
then the solution is heated water bath
until it reaches 60 degrees Celsius. The
garlic was next minced and weighed up
to 25 g and then added to the
homogenizing solution, for 5 minutes the
solution should be heated and stirred.
After the water bath 1.5 grams of papain
or 4 ml of meat tenderizer was added to
the solution then water bath, 60 degrees
Celsius, for another 10 minutes.
After water bath the flask should be in
an ice bath for 5 minutes. The solution is
swirled for it to cool evenly. This process
will impede the breakdown of the DNA
by deoxyribonucleases. After it cool, it
was poured in a blender, for
45 seconds for it to homogenized. With
the pipette 15-20 ice- cold ethanol is
added to the mixture. allowing it to
dripped down at the side of the tube.
The ethanol formed on top of the onion
filtrate.
2. Ultraviolet Measurement of Isolated
DNA
For high volume determination 0.5 ml of
the aliquot solution was dissolved in a
4.5ml TE buffer, or dilute with the ratio of
1:20. After dilution the solution is
transferred to the microplate for the
determination of absorbance (at
260,230 and 280).
3. Acid Hydrolyisis
4. Test for Deoxyribose
The 3.5 diphenylalanine reagent was
added nto the 1.5 hydrolyzed DNA and
repeated with the 0.5-ml standard
deoxyribose solution, then the both
tubes was placed in a boiling water
bath for 10 minutes then cooled
5. Murexide test (test for purines)
In a small evaporating dish with 5-10
drops of nucleic acid solution fes\w
drops of concentrated nitric acid was
added. After addition the solution was
evaporated carefully until dry, then the
residues were moistened with 10%
potassium hydroxide the n heated. After
heating the solution was tested again
with potassium hydroxide then heated
again . after heating a few drops of
water was added then warmed. Lastly,
the residue was evaporated again. The
 process was repeated for guanine and CONCLUSION
adenine

6. Wheeler-Johnson test (test for

pyrimidines)
The 0.5 ml of nucleic acid was treated
with excess bromine water until solution
turned to yellow, it must be repeated
with cytosine and uracil, after treating
the solution was boiled, to remove the
excess, the solution should be colorless
or light yellow. Then barium hydroxide REFERENCES
was added to the solution. Lastly test
the solution with a litmus paper. Anthony Carpi, Ph.D. Nucleic Acids
. Visionlearning Vol. BIO-1 (1), 2003
retrieved 2011 from:
http://www.visionlearning.com/en/library/
Biology/2/Nucleic-Acids/63
RESULTS AND DISCUSSION Mc-Graw Hill DNA: the genetic
,material from:
Table 1. test for purines results https://highered.mheducation.com/sites/
9834092339/student_view0/chapter14/d
Results na_structure.html
Nucleic acid Red
solution
Adenine Yellow
Guanine Red

Table 2 . test for pyrimidines result

Results
Nucleic acid Yellow
solution
Cytosine Violet/ Purple
urcil Purple