EXPERIMENT 1

COLORIMETRIC ESTIMATION
OF DNA BY
DIPHENYLAMINE
METHOD

Structure
1.1 Introduction 1.5 Precautions

Expected Learning Outcomes 1.6 Summary

1.2 Principle 1.7 Self Assessment Questions

1.3 Material Required

1.4 Protocol

1.1 INTRODUCTION
DNA is deoxyribose nucleic acid that carries the genetic information in a cell
and is capable of self replication and synthesis of RNA. DNA consists of two
long chain of nucleotides twisted into a double helix and joined by hydrogen
bonds between the complementary bases adenine and thymine or guanine
and cytosine. DNA acts as a genetic material and carries the genetic
information. The sequence of nucleotides determines individual hereditary
characteristics.

As you know, nucleotides are the building blocks of all nucleic acids.
Nucleotides have a distinctive structure composed of three components
covalently bound together: Nitrogen-containing "base" - either a pyrimidine
(one ring) or purine (two rings), five-carbon sugar - ribose or deoxyribose, and
a phosphate group (Fig. 1.1). The combination of a base and sugar is called a
nucleoside. Nucleotides also exist in activated forms containing two or three
phosphates, called nucleotide diphosphates or triphosphates respectively. If
the sugar in a nucleotide is deoxyribose, the nucleotide is called a
deoxynucleotide; if the sugar is ribose, the term ribonucleotide is used.

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BBCCL-118 Gene Organisation, Replication and Repair

Fig. 1.1: Structure of a nucleotide.

The bases of DNA and RNA are heterocyclic (carbon and nitrogen-containing)
aromatic rings. Adenine (A) and guanine (G) are purines, bicyclic structures
(two rings), whereas cytosine (C), thymine (T) and uracil (U) are monocyclic
pyrimidines. In RNA, the thymine base is replaced by Uracil (U) (Fig. 1.2).
DNA is an important biological molecule, which can be isolated from various
sources. To characterize the DNA sample it is often necessary to determine
its concentration. The concentration of a DNA sample can be determined
using diphenylamine method using a colorimeter or a spectrophotometer. In
this method a set of standards (where the concentration of DNA is known) is
used and the concentration of the unknown sample is then determined by
comparing it with the standards.

Fig. 1.2: Components of nucleic acids.

Expected Learning Outcomes

After studying and performing this experiment, you should be able to:

 understand the principle of DPA (Diphenylamine ) reaction with DNA;

and

 perform the experiment to determine the concentration of DNA in a given

solution.

1.2 PRINCIPLE
Diphenylamine reaction is not specific for DNA. It is a reaction given by
2-deoxypentose sugar. As the DNA contains 2-deoxypentose sugar, this
6 reaction can be utilized for the estimation of DNA. When DNA is treated with
Experiment 1 Colorimetric Estimation of DNA by Diphenylamine Method

diphenylamine reagent under acidic condition, the purine nucleotides are

released from the DNA and the 2-deoxypentose sugar of the purines reacts
with acid to form ω-hydroxylevulininic aldehyde which in turn reacts with
diphenylamine reagent to form a blue colored complex (Fig. 1.3) with
absorbance maxima at 595 nm. Here you must note that, in DNA, only the
deoxyribose of purine nucleotide reacts so that the value obtained represents
one half of the total deoxyribose produced. However, as the readings are
compared with standard DNA, which also reacts in a same way, so the
concentration of DNA can be determined accurately.
The amount of blue
coloured complex is
proportional to the
concentration of DNA.

Fig. 1.3: Reaction of Diphenylamine with DNA.

1.3 MATERIAL REQUIRED

Equipment

 Colorimeter or Spectrophotometer

 Water bath

Chemicals/reagents

 Standard DNA solution

 Diphenylamine reagent

 Glacial acetic acid

 Conc. H2SO4

 Distilled water

 Acetaldehyde

 Test Sample of DNA 7

BBCCL-118 Gene Organisation, Replication and Repair

Glasswares and others:

 Test tubes

 Pipettes

 Graduated cylinder

PREPARATION OF REAGENT:

1. Standard DNA solution (1mg/ml): Dissolve 10 mg of calf thymus DNA in

10 ml of 1N HClO4 by heating at 70oC for 15 minutes.

2.1.6% Acetaldehyde solution: Dissolve 1ml of ice cold acetaldehyde in 50

ml of distilled water.

3. Diphenylamine solution: Dissolve 1.5 g of diphenylamine in 100 ml of

glacial acetic acid and 1.5 ml of conc. H2SO4.

4. Diphenylamine reagent: Add 0.5 ml of 1.6% of acetaldehyde into 100 ml of

diphenylamine solution.

1.4 PROTOCOL
1. Take 9 test tubes and label them as B, S1, S2, S3, S4, S5, S6 and U1
and U2 (where B = blank, S = standard and U = unknown).

2. Pipette different volumes of standard DNA solution and water according

to the protocol Table 1.1. In the unknown tube take 0.5 and 1 ml of DNA
sample whose concentration we have to determine.

Table 1.1: Protocol Table

 Clear test tube S. Standard Unknown Distilled Diphenylamine

indicates no No. DNA DNA Water Reagent (ml)
nucleic acids. solution solution (ml)
 Blue colour (1mg/ml) (ml)
indicates the
presence of DNA. B 0 --- 1 4
 Greenish colour S1 0.2 --- 0.8 4
indicates the
presence of RNA. S2 0.4 --- 0.6 4

S3 0.5 --- 0.5 4 Heat the

tubes in
S4 0.6 --- 0.4 4 boiling
water bath
S5 0.8 --- 0.2 4
for 15
S6 1.0 --- 0 4 minutes

U1 --- 0.5 0.5 4

U2 --- 1.0 0 4

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Experiment 1 Colorimetric Estimation of DNA by Diphenylamine Method

3. Add 4 ml of diphenylamine reagent to all the tubes and heat the tubes in
boiling water bath for 15 minutes.

4. Cool down the tubes and take the absorbance at 595 nm by setting the
blank with blank tube.

5. Record your observations in Table 1.2.

6. Draw a standard curve with the amount of DNA (µg) at x axis and
Absorbance at 595nm at y axis. From the graph calculate the amount of
unknown DNA sample.

Table 1.2: Observation Table

S.No. Amount of DNA (in µg) Absorbance at 595 nm

S1

S2

S3

S4

S5

S6

U1

U2

1.5 PRECAUTIONS
1. Make diphenylamine reagent fresh.

2. Instead of heating, the tubes can also be incubated at room temperature

in dark for 16-18 hrs before taking absorbance.

3. If the absorbance of the unknown sample is not falling within the

standard tubes values further dilute the sample or if it very low then
dissolve the DNA extracted from a source in smaller volume.

4. If the reading of standards is falling out of range of spectrophotometer

then dilute the standard solution and make the standard tubes again.

5. Handle the test tubes carefully as the tubes contain DPA reagent which
has acid in it.

6. Diphenylamine is harmful so handle it carefully.

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BBCCL-118 Gene Organisation, Replication and Repair

1.6 SUMMARY
DPA reaction was used for estimation of DNA concentration in the given DNA
sample. DNA reacted with DPA along with the standard DNA samples and the
absorbance was taken at 595 nm. The readings were plotted on the graph and
from the graph the concentration of DNA sample was determined.

1.7 SELF ASSESSMENT QUESTIONS

1. What are the different components of DNA?

2. Define and draw the structure of a nucleotide.

3. Explain the principle behind use of DPA for estimation of DNA.

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