ISOLATION AND QUALITATIVE COLOR REACTIONS OF DNA

Cabantog, Rachel Joy R., Cadacio, Alyzza C., Castellano, Michael Justin C.
Catipay, Marielle Anne B., Chin, Jan Jason C.
Group 3, 2D Medical Technology, aculty o! "har#acy, $ni%ersity o! &anto To#as
ABSTRACT
Deoxyribonucleic acid (DNA) is one of the essential biochemical molecules of an organism that serves as the
repository of genes. The objectives of the experiment are to isolate DNA from the onion (Allium cepa) sample and
to perform qualitative color reactions. DNA as isolated from onion by disrupting its cell membrane and other
cellular components. !rom the experiment" the isolate as found to have a thread#li$e structure. The sample as
subjected to qualitative color reaction tests that such as test for deoxyribose" test for phosphate" test for
pyrimidine and test for purines. !or the results" the hydroly%ate produced blue solution for test for deoxyribose
hich is a negative result& yello solution ith hite precipitate for test for phosphate hich is also a negative
result& and yello residue for test for purine hich indicates the presence of adenine hoever" it produced a hite
solution for test for pyrimidines hich is a negative result.
INTRODUCTION
Nucleic Acid is one of the essential biochemical
molecules present in an organism. It function for
the encoding, transmitting, and expressing of
genetic informations. It has two types namely the
DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid).
Fig. 1 The structure of nucleic acid
Deoxyribonucleic acid is a nucleic acid that
contain genetic instructions used in the
development and functioning of all $non living
organism. DNA is made of chemical building
bloc$s called nucleotides. These building bloc$s
are made of three (') parts& phosphate group"
sugar group" and one of four types of nitrogen
bases. DNA strand is formed by the lin$ing of
nucleotides into chains ith a phosphate and
sugar group in an alternating manner.
The four nitrogen bases found in nucleotides
are( adenine (A)" thymine (T)" guanine ())" and
cytosine (*). The order or sequence of these
bases determines hat biological instructions
are contained in a strand of DNA.
Fig. 2 The structure of DNA
D'A contain the instructions needed !or an
organis# to de%elop, sur%i%e and reproduce. To
carry our those !unctions, D'A se(uences #ust be
con%erted into #essages that is used to produced
proteins, )hich are the co#ple* #olecules that do
#ost o! the )or+ in our bodies.
EXPERIMENTAL
A. Compounds used: +nion" homogeni%ing
solution" papain (or meat tenderi%er solution)"
ice#cold ethanol" T, buffer" diphenylamine"
*onc. -./+0" conc. -N+'" 123 (N-0).4o+0
solution" 123 5+-" 6romine ater" 6a(+-)..
B. Procd!r"
,. -solation o! D'A !ro# onion
A ./#0 ho#ogeni1ing solution is prepared in
a ,2.#0 2rlen#eyer !las+ and )as heated in a
)ater bath until the solution reached 3/ 7*" .8g
#inced onion )as added to the preheated )ater
bath. ,..g papain )as then added and +ept !or
,/ #inutes. A!ter heating, place the 2rlen#eyer
!las+ to an ice bath !or about . #inutes, then
place the solution in a bea+er to be
ho#ogeni1ed. A!ter ho#ogeni1ation, the
ho#ogenate )as then !iltered using !our4
layered cheese cloth into a 2./#0 bea+er and
cool a!ter )ards. The cooled solution )as added
)ith ,.42/#0 o! ice4cold ethanol in the side o!
the bea+er allo)ing it to drip slo)ly to the
ho#ogeni1ed solution. D'A )ill initially
precipitate out !or 34. #inutes )ithout
disturbing because it not soluble in ice cold
ethanol. &pool the !or#ed D'A using pre4bent
glass pipette and sub5ect speci#en )ith T2
bu!!er solution
2. 6ualitati%e color reactions
a# T$% &or Do'yri(o$
Add 3..#0 diphenyla#ine reagent to
,..#0 hydroly1ed D'A solution. The sa#e
procedure is done to /..#0 standard
deo*yribose solution. "lace the #i*ture in
hot )ater bath !or at least ,/ #inutes then
cool i##ediately and obser%e results.
(# T$% &or P)o$*)a%
Add 1m9 of conc. -./+0 to 1m9 nucleic
acid solution and heat in a small flame and
mix until the contents turned bron. *ool
off and add 2.8m9 conc. -N+' then heat
again until hite fumes appear and turn
colorless. Add 1m9 of -.+ to the colorless
liquid then heat in a boiling ater bath.
After ater bath" cool it off and add 1m9
123(N-0).4o+0 solution. 4ix the solution
and dilute ith 12m9 -.+. 9et solution
stand for 8 minutes. Ta$e note of the
formation of the precipitate.
c# T$% &or P!ri+$
"lace ,/ drops o! nucleic acid solution into
a s#all e%aporating dish then add !e) drops
o! 7'8
3.
Care!ully e%aporate until dry in a
)ater bath. Moisten the e%aporated nucleic
acid )ith ,/9 :87 and heat !urther, note
the color changes upon adding the :87 and
upon heating. Add !e) drops o! 7
2
8 then
obser%e the color. 0astly, e%aporate and note
the color changes.
d# T$% &or Pyri,idi+
Treat /..#0 nucleic acid )ith e*cess
bro#ine )ater until solution turns yello).
Re#o%e e*cess by boiling the solution until
it turns yello). 'ote the changes, add e*cess
Ba;87<
2
solution then test )ith lit#us paper.
Ta+e note o! the appearance o! the solution.
RESULTS AND DISCUSSION
-. I$ola%io+ o& DNA &ro, o+io+
-n ho#ogeni1ation, the onion )as
#inced then heated. The heat treat#ent is
done in order to so!ten the cell )all and !or
the brea+do)n o! the cells. 7eating so!tens
the phospholipids in the cell #e#brane
and denatures the ribonuclease en1y#es,
)hich i! present can cause !rag#entation
o! the D'A and thus pre%ent it !ro# being
spooled.
.. Q!ali%a%i/ Color Rac%io+$
C),ical T$% DNA0o+io+# DNA0$%a+dar
d#
Do'yri(o$ Bl! $ol!%io+ Bl! $ol!%io+
P)o$*)a% 1llo2
$ol!%io+ 2i%)
2)i% **%.
1llo2 **%.
Pyri,idi+ 3)i%
$ol!%io+
P!r*l
$ol!%io+
P!ri+ 1llo2
r$id!
1llo2 %o rd
r$id!
Table 1. Results for color reactions
As sho)n in the table, there are %arious results
!or the color reactions o! D'A.
a) Test for deoxyribose:
he reaction between the dischei reagent and !"
deoxy pentose results in the de#elopment of a blue
color. he reaction depends on the con#ersion of the
pentose to hydroxy lae#unilic aldehyde which then
reacts with diphenylamine to gi#e a blue colored
complex. he intensity of the blue color is
proportional to the concentration of the DNA. he
sample from onion and standard solution produced
a positi#e result.
b) Test for phosphate:
Ammonium molybdate reacted with the sample
which yields yellow crystals forming
phosphoammonium molybdate which is a positi#e
result. $ut in the sample, it only produced yellow
solution and white ppt. which means it is negati#e in
this test. %or the standard solution, it yielded yello
precipitate indicating that it is positi#e in this test.
c) Test for purines:
DNA reacted with nitric acid since purines is &nown
to be readily soluble in diluted acid. Nitric acid
oxidi'ed it lea#ing a yellow precipitate. he sample
from both onion and standard solution produced a
yellow residue which indicates a negati#e result.
d) Test for Pyrimidines:
$romine water reacted with the sample to form ("
bromo" )" hydroxyhydroxo deri#ati#e which pro#ides
a green coloration. *pon addition of $a(+,)! will
gi#e a result of purple precipitate. he sample from
onion produced a white solution which is a negati#e
result while on the other hand, the standard sample
yield a purple solution which is also a negati#e result.

REFERENCES
ro# internet=
:ualitative Testing for Nucleic Acids
http(;;labopslton.i$ispaces.com;fil
e;vie;:ualitative<Testing<for=Nucleic
Acid<3.><?roteins.pdf
Nucleic Acid Tests.
http(;;forum.daffodilvarsity.edu.bd;
index.php@topic=AA>B.2
%rom boo&s-
Mathe)s, C., %an 7olde, :.2. ;,>>3<
Biochemistry. The Ben5a#in?Cu##ings
"ublishing Co#pany, -nc.