..

Quantitative estimation of DNA by ~

the diphenylamine
.':,

reaction

PRINCIPLE
o~"-
Under extreme acid conditions, DNA/is initially depurinated quantitatively
followed by the dehydration of sugar to i-
hydroxylevulinaldehyde. This aldehyde
condenses, in acidic medium, with diphenylamine to produce a deep blue colored
condensation product with absorption maximum at 595 nm. This reaction is given
by _2-deoxypentoses in general and is not specific for DNA. In DNA, since only
the deoxyr1bose of the purine nucleotides reacts, the total value obtained
represents h~lf of the total deoxyribose present.

. I
MATERIALS

Chemicals
(\
1. DNA (Commercial sample) 20mg
2. Buffered saline 100ml
3. Diphenylamine 10 g
· 4. Glacial acetic acid A.R. 1 litre
5. Cone. sulphuric acid A.R. 25ml

Equipment
Boiling water bath
Spectrophotometer/Colorimeter

PREPARATION OF SOLUTIONS

1. Buffered saline pH 7.0

(Mo!.wt. of NaCl 58.44, Mol. wt. of tri-sodium citrate dihydrate 294.1 O) No t,
!o ~r~pare buffered. saline, dissolve 8. 77g of NaCl and 4.41 g of Sodium citrate
in d1st1lled water. Raise the volume to 1 litr~ with distilled water.

2. Diphenylamine reagent
To pre~are di~hen~lamine reagent, dissolve 1Og of pure di phenyl amine in 1 litre
of glacial acetic acid and add 25 ml of concentrated sulphuric acid. This sol f
must be prepared fresh. u ion

METHOD

1. Dissolve 20mg DNA in 100ml of b4ffered saline i.e 200 ~

. 2. In a set of test tube~, add 0-400~ g of DNA ( 0.0ml, 0.4 :r,7r,·
:o.r ·
I

1.6ml and1ct~ 9f.11). AdJust the volume to 2.0 ml with buffered saline
o~a
:J1:
·
/i°~i
'
· Add 4.0 ml of aiphenylamine reagent. Mix thoroughly. ·

44
Re.Q.J?{M
l)NA._ 11 <.{ ' Oli :, Nwe,ocrclL t>U Pl,w6~1,,_~ 0

k p16.,r"'M-'°"'e. o.Mt1 pwdtit. ~r&J..

• • • .6,_ , .· . I •;, ! . " . > I

1-1?,P011
'v
o~, , - ~ ~ ' . Ji
"'c...-
. , .I . 'c/
.I .. ,

_;. -o-~-o
H
CJ-<1- - . • • CM 2-
\ 1-1~o•
_,-~~'>- 11 '
- c.--0!'1 (,,!A, 1,
' ~sfu ·lblo<:t,,
I I
1-1 --c.. ..... ol-'I c.,. ;=-? ·C..0(1'lp·I~
I I
(.11i.,0"1.•· ' ,C ,),\i.-0~

. rl-- ½d-r~ .

CoJ wJ 0-hooYl__ g-
.)0 °=f DNI/ f71 /oo ri4.. 1 h,uff-u-< /2a1Jne.
1 ?¾l. of 6olu-h· <>1. :=_ . «0 1, DN 4
/00 J
..
4,.,cw,J- aj 6/oc.t ,DNA ;;. 0.2 Mj °f- bf!!1_
in l Mi 1 ~o(,u,/-ier, -=- .. t>JOO "43 /~ ·. ~NA•
I ,, \

('101..U, Q.A.,,,1oW-A-f 1- DNFJ l U,M)u,,wu]'"1) = pv..1..(,l'eM.,t_ + p'f'rHrcJ.Jne_

:z o> " .ioo~ I M1,
., 1.100 ~ - /Mf_

\,) i,., t- (.IIYl)CHl.b W"1 b"' A ;o . ~ . - J

I
 Scale: X-axts:1 cm- 20ug
Y-axts:1cm•O.OSOO

.. 1------...,.-~,,.---t
si " < - - - - -~~,,.-----I
.. " t---.,,,,~- ~,,.,,-------l
',,,,.
" 1-...-~---------l
l , S 5 7 I 1 10 II
Coec:-tnDN ol DSA ill . .
4. Place a marble at the mouth of the tube and heat in a boiling water bath for
10 minutes. Cool. ·
5. Read the absorbance at 595nm for DNA content of each tube and calculate
net absorbance.
6. Plot the standard graph between net absorbance and DNA concentration.
7. Repeat the steps for given DNA sample of unknown concentration.
8. Estimate the concentration of unknown sample from the standard curve.

PRECAUTIONS

1. Handle diphenylamine reagent carefully since it is mutagenic and toxic.

REFERENCES

1. Plummer D.T.1979. An Introduction to Practical Biochemistry. Tata McGraw-

Hill Publishing Company Ltd.
2. Sadasivam S. and Manickam A. 1992. Wiley Eastern Limited.
3. Sawhney S.K. and Singh R. 2000 Eds. Introductory Practical Biochemistry.
Narosa Publishing House.

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