Diphenylamin

e Test for
Deoxyribose

By: Anne Marielle D.

Manalo
Object
ive:
To detect the
presence of
deoxyribose
in DNA.
Procedure:
1. Set up 4 dry and clean test tubes. Add 2 ml of
each of the following:

1 Test
tube:
st

2 Test
tube:
nd

3 Test
tube:
rd

4th Test
tube:
Crude
Procedure:
2. Add 5 ml of diphenylamine solution to each test tube.
Mix the contents of the test tubes.
Procedure:
3. Heat the test tubes in a boiling water bath for
10 minutes. Observe the color of the solution.
Result
s:
Test
tube
1
2
3
4

Solution

Observations

1% glucose Brownish black

1% ribose Brownish black
1%
Light blue
deoxyribose
Crude DNA Brownish black
solution
Principle:
The
DNA
is
treated
with
diphenylamine
under
acidic
condition
and
is
initially
depurinated
quantitatively
followed by the dehydration of
deoxysugar
to
ωhydroxylevulinylaldehyde.
This
aldehyde condenses in an acidic
medium with diphenylamine to
produce
deep
blue
colored
solution.
Sample Equation Involved:
Explanation of Results
DNA and RNA are nucleic acids made
of nucleotide subunits. One major
difference between DNA and RNA is
their sugar: DNA contains deoxyribose,
whereas RNA contains ribose.
Deoxyribose
is
a deoxy
sugar,
meaning that it is derived from
the sugar ribose by
loss
of
an oxygen atom. Since it is DNA that
contains deoxyribose, therefore the
standard DNA and crude DNA solution
will show positive result.
The intensity of the blue
color is proportional to
the concentration of DNA.
It increases along with
the
increase
concentration of DNA.
How does one minimize the
interaction between the DNA and
histones in the experiment?
Proteins and water-soluble molecules will stick to the
DNA until we add a sodium chloride solution—this
will minimize the interaction between positivelycharged protein molecules and
negatively-charged
DNA. The addition of ethanol then separates the
large molecules (DNA being the largest of the
molecules in solution) from the smaller molecules
(protein and RNA). The DNA will form a thread-like
precipitate that can be spooled onto a rod, while the
gelatinous mixture of proteins and RNA are left
behind. This crude extract of DNA will still contain
some contaminant proteins, but it is mostly DNA.
What are histones?
Histones are positively charged proteins
that facilitate the packing of DNA into
condensed chromatin fibers. They have
many arginine and lysine amino acids
that easily bind to the negatively
charged DNA. The double helix of DNA is
highly negatively charged due to all the
negatively charged phosphates in the
backbone. All that negative charge must
be counterbalanced by a positively
charge proteins, histones, that bind DNA
and aid in DNA's packaging.
Modifications of Histones
Normally, histones are
positively
charged
molecules, and addition
of
methyl
groups
(methylation)
makes
them more hydrophobic.
Hydrophobic molecules
tend to stick together,
and increasing histone
methylation will cause
the histones to pack
Modifications of Histones
Acetylation (adding an acetyl
group)
and
phosphorylation
(adding a phosphate group) make
the
histones
more
negatively
charged
because
acetyl
and
phosphoryl groups are negative. By
making histones more negatively
charged, their grip on DNA will be
much looser because DNA is also
negatively charged. Similar charges
(negative and negative) repel one
another.