Diphenylamine Test

for Deoxyribose
By: Anne Marielle D. Manalo
 Objective:

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:

1stTest tube: 2nd

Test tube: 3rdTest tube: 4th Test tube:
1% glucose 1% ribose 1% deoxyribose Crude DNA
solution
 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.
Results:
Test tube Solution Observations
1 1% glucose Brownish black

2 1% ribose Brownish black

3 1% deoxyribose Light blue

4 Crude DNA solution Brownish black

 Principle:
The DNA is treated with
diphenylamine under acidic condition and
is initially depurinated quantitatively
followed by the dehydration of
deoxysugar to ω-hydroxylevulinylaldehyde.
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. The
darker the color the greater the
concentration of DNA in the solution.
 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 positively-charged 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 even more
tightly than usual.
 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.