EXPERIMENT 8

Hydrolysis of Nucleic Acids

Group 8
COMILANG
NASSER
SUNUR
Objectives  To prepare hydrolyzed
samples of DNA and RNA
 To differentiate between acid-
hydrolyzed, base-hydrolyzed,
and unhydrolyzed nucleic
acids (DNA & RNA) through
the qualitative tests employed
 To identify purity of RNA and
DNA isolated from yeast and
onion, respectively
 Hydrolysis of nucleic acids
NTRODUCTIO  chemical or enzymatic
 oligonucleotides, nucleosides, ribose or deoxyribose,
phosphates

 Acid hydrolysis
•susceptible: purine N-glycosyl bonds
•resistant: pyrimidine N-glycosyl bonds

RNA
 boil with dil. acid  adenine and
guanine and pyrimidine nucleotides
 heat pyrimidine nucleotides in an
autoclave or sealed tube  cytosine
and uracil
 treatment with 1M HCl at 100° for 1
hour
 DNA (acid hydrolysis)
NTRODUCTIO -treatment at pH 3.0 : removes purines

Base hydrolysis
RNA
dilute NaOH or KOH
obligatory intermediate: cyclic 2',3‘-
monophospates
attack on one P-O-C linkage : 2' and 3'-
nucleoside monophosphates

DNA
 nucleases
sugar 2° structure
phosphodiester bond bases
 Preparation of:
NTRODUCTIO  Acid and Base Hydrolyzed DNA and RNA
samples
 Unhydrolyzed DNA and RNA samples

Tests
 Benedict's test
 Orcinol/Bial’s Test
 Test for Purine bases
 Test for Inorganic Phosphate
 Test for Protein
 Test for Deoxyribose
 Test for A & G
 Test for U & C
Preparation of Samples
I. Acid Hydrolysis of DNA/RNA
Test tube:
Cover with
RNA/DNA
marble *Add
sample HYDROLYSATE!
Boiling water
10mL 10%
water bath
H2SO4
for 1hr

II. Alkaline Hydrolysis of DNA/RNA

Test tube: Cover with marble
*Add
RNA/DNA sample Boiling water bath
water
5mL 10 NaOH for 1hr

Cool hydrolysate
Add glacial acetic HYDROLYSATE!
acid (pH 4-6)
Preparation of Samples III. Solution of RNA

RNA sample 10 mL RNA

0.05 % solution!
NaOH

IV. Solution of DNA

DNA 10 mL DNA
sample distilled solution!
water
 Benedict’s Test
Qualitative
• Test for REDUCING SUGARS (i.e. aldehyde
& ketone group)
• Neutralize 5 drops of the hydrolysate
Tests
samples with solid Na2CO3; decant!
• Control : 0.1 % glucose
2 drops of
Boiling
the samples Benedict’s
water OBSERVE!
in separate reagent
bath
test tubes

• Expected : green, red, yellow precipitate

• Ribose : w/ aldehyde in linear form
• Deoxyribose : aldopentose; w/ aldehyde in
linear form
 Benedict’s Test
Qualitative
Tests

CYCLIC CYCLIC RIBOSE

DEOXYRIBOSE

ACYCLIC RIBOSE
*sugars occur in a chain as well as a ring structure; possible to have an equilibrium between two
forms. When the hemi-acetal or ketal hydroxyl group is free or not linked to another molecule, the
aldehyde (or ketone) form is available for reducing copper (II) ions. When a sugar is oxidized, its
carbonyl group (i.e. aldehyde or ketone group) is converted to a carboxyl group.
 Benedict’s Reagent
Qualitative
• Contains Cu2+ as copper sulfate (CuSO4)
• Cu2+ react with reducing sugars = precipitate
Tests
of cuprous oxide (Cu2O) produces a change
in the Benedict’s reagent from blue to
green or reddish-orange, depending on
amount of reducing sugar.
• Green = small am’t of reducing sugars
• Red-orange = abundance of reducing sugars
 Benedict’s Test
Qualitative
Tests

CONTROL ACID
HYDROLYZED
RNA
 Bial’s Test
Qualitative
• Test for PENTOSES
• Control : 0.1% xylose (0.1% arabinose or
ribose)
Tests
2 drops
unhydrolyzed DNA &
RNA samples; with 1 4 drops
boiling water
drop 12 M HCl Orcinol
bath
2 drops of DNA and reagent
RNA hydrolysate
• specimen is heated with sol’n of orcinol,
HCl, and FeCl3
• pentoses : dehydrated to furfural = reacts
with orcinol to form a green product
 Bial’s Test
Qualitative
Tests
FURFURAL

CONTROL

RNA SAMPLES : U A B
 Test for Purine
Bases
Qualitative

6M NH3
5 drops Leave Observe the
until alkaline
sol’n crystals
Tests
of each
sample Few drops of untouched under the
5% AgNO3 ‘til crystals microscope
form

• Flocculent, gelatinous white

precipitate
flocculation  suspension of particles
that coalesce, or flocculate, during the
sedimentation process
Qualitative Test for Purine Bases
Tests

RNA SAMPLE : U A B

DNA SAMPLES : U A B
Qualitative Test for Purine Bases
Tests

RNA U, FRESH RNA A, FRESH RNA B, FRESH

RNA U, DRIED RNA A, DRIED RNA B, DRIED

 Test for Inorganic
Phosphate
Qualitative
• Control : Na2HPO4
Excess 5 drops
5 drops ammoni ammoniu Warm and
Tests
of each a m allow to
sample Acidify molybdat stand
with 6M e reagent
HNO3

• Expected : yellow ammonium

molybdophosphate precipitate; yellow color
followed by yellow ppt
• Heating : hydrolyze pyrophosphate to phosphate
HPO4 2-
(aq) + 12MoO4 (aq) + 3NH4 (aq) + 23H3O
2- + +
(aq)
 (NH4)3[P(Mo3O10 )4](yellow,s)
Qualitative Test for Inorganic Phosphate
Tests

CONTROL DNA U DNA A DNA B

RNA U RNA A RNA B

 Test for
Deoxyribose
Qualitative
• “Dische Diphenylamine Test”.
• Expected: blue solution
Tests
• Conversion of pentose to ω-
hydroxylaevulinic aldehyde; reacts with
diphenylamine = blue colored complex
• Intensity of blue color : proportional to
DNA conc.
2 ml Boiling
5 ml
portion water OBSERVE!
diphenylami
of bath for
ne reagent
sample 5min.
Qualitative Test for Deoxyribose
Tests
Qualitative Test for Deoxyribose
Tests

DNA SAMPLE : U A B

RNA SAMPLES : U A B
 Test for Uracil and
Cytosine
Qualitative
• “Wheeler and Johnson's test”
Tests
1 ml 1 ml
BOIL! Excess
sampl bromine
Ba(OH)2
e sol’n water

• Treatment w/ bromine water yields

dialuric acid = green coloration
• Addition of Ba(OH)2 = purple.
 Biuret Test
2 ml 10%
Qualitative
1 ml NaOH sol’n
sample MIX
sol’n 5-10 drps WELL
0.5% CuSO4
Tests
sol’n
• Forms chelate* w/ an intense violet-red color
w/ the Cu2+ ions in alkaline sol’n
• Positive : tripeptides and polypeptides
• Negative for : dipeptides or free amino acids;
may result in a pinkish color

* A chemical compound in the form of a heterocyclic ring, containing a metal ion

attached by coordinate bonds to at least two nonmetal ions.
Qualitative Biuret Test
Tests

DNA SAMPLE : U A B

RNA SAMPLES : U
 FORMATION OF PURPLE COMPLEX
Qualitative
Tests
 Xanthoproteic
Test
Qualitative
Nitration of an aromatic ring
Phenyl rings : nitrated to produce a
Tests
yellow product  test for the
presence of Phe, Tyr, Trp
Strong base = orange.
Certain benzene compounds also
give a positive test
Tyrosine = yellow; tryptophan =
orange
 REACTION WITH NITRIC ACID
Qualitative
Tests
 Test for Adenine and Guanine
Qualitative
• “Murexide Test”
• Murexide (NH4C8H4N5O6, or C8H5N5O6.NH3) or
ammonium purpurate [MX] = ammonium
Tests
salt of purpuric acid.
• Test for presence of uric acid = white,
odorless, tasteless crystalline substance
from purine degradation
• Positive : yellow residue
• A & G : purines = 2-ringed crystalline
organic base – a uric acid; heterocyclic
aromatic org. compound composed of
pyrimidine ring fused w/ an imidazole ring
 Test for Adenine and Guanine
Qualitative
Tests

URIC ACID (7,9-dihydro-1H-

purine-
2,6,8(3H)-trione)

MUREXIDE (Ammonium 2,6-dioxo-5-[(2,4,6-

trioxo-5-hexahydropyrimidinylidene)amino]-
3H-pyrimidin-4-olate)
 1. How can intact DNA be obtained from a

Questions solution of DNA and RNA?

• Alkaline hydrolysis
• DNA not readily hydrolyzed : no 2’-
OH group; cannot form necessary
2’,3’-cyclic monophosphate
intermediate
• RNA hydrolyzed by dilute alkali to
produce intermediates : 2’,3’-cyclic
monophosphates; hydrolyzed  2’-
& 3’-nucleotides; further hydrolyzed
 2’- & 3’-nucleoside
monophosphate (attack either one
of two P-O-C linkages)
 2. Based on the qualitative tests, how can

Questions you differentiate hydrolyzed from

unhydrolyzed RNA?
• Unhydrolyzed RNA: positive for all except
for the presence of uracil and cytosine
 Pentoses, purine(A & G) and pyrimidine bases,
proteins, and phosphoric acid were extracted
 No uracil
• Acid-hydrolyzed RNA:
 Trace amounts of reducing sugars, pentoses,
purine(A & G) and pyrimidine bases, Phe, Tyr, Trp,
and phosphoric acid
 No tri- and polypeptides
• Base-hydrolyzed RNA
 pentoses, purine(A & G) and pyrimidine bases, tri-
and polypeptides, Phe, Tyr, Trp, and phosphoric
acid
0.05% NaOH is the best reagent to
separate RNA into its components
 3. Based on these experimental results, do

Questions you think you obtained pure RNA from

yeast? Why or why not?

• We obtained impure RNA

from yeast
Pentoses present : RNA and/or DNA
Deoxyribose not present : RNA
Uracil and Thymine : not present
Purine bases are present
Presence of tri- and polypeptides =
impurities
 4. Based on these experimental results, do

Questions you think you obtained pure DNA from

onion? Why or why not?

• We obtained pure DNA from

onion (for acid-hydrolyzed
sample)
Trace amounts of deoxyribose
Adenine and guanine test positive
No cytosine
No tri- and polypeptides
Positive test for Xanthoproteic =
may signify presence of other
benzene ring derivatives
 • Acid hydrolysis of DNA extracts trace amounts of pentoses
(deoxyribose), purine bases (A & G), and proteins from DNA =
Conclusio
good way to separate DNA into its components
• Base hydrolysis of DNA cannot extract any of the components
being tested.
• Unhydrolyzed DNA : negative for all except for Xanthoproteic
test and test for inorganic Pi
• Acid hydrolysis of RNA extracts trace amounts of reducing
sugars, pentoses, purine bases, proteins, and phosphoric acid.
• Base hydrolysis of RNA extracts pentoses, purine bases,
ns
proteins, and phosphoric acid.
• Unhydrolyzed RNA positive for all test except presence of C & T;
and A & G = best way to separate RNA into its components.
• Sample extracted from yeast contains RNA, but impure.
• Sample extracted from onion contains pure DNA.
 • http://apple.cmu.edu.tw/~a001003/Carbohydrates%20-
%20Qualitative%20Tests.htm
•
References http://en.wikipedia.org
• http://faculty.mansfield.edu/bganong/biochemistry/lipids3.htm
• http://faculty.mansfield.edu/bganong/biochemistry/proteins.htm
• http://focosi.immunesig.org/invitrocellfree.html
• http://library.med.utah.edu/NetBiochem/pupyr/pp.htm
• http://members.fortunecity.com/badudz/experiment9.html
• http://syncope.blog.com/612609/
• http://www.cerlabs.com/experiments/10875404472.pdf
• http://www.drugs.com/dict/wheeler-johnson-test.html
• http://www.frtr.gov/matrix2/section4/4-50.html
• http://www.mercksource.com/pp/us/cns/cns_hl_dorlands_split.jsp?
pg=/ppdocs/us/common/dorlands/dorland/eight/000107483.htm
• http://www.rowan.edu/biology/Labs/biomole.pdf
• http://www.saddleback.edu/faculty/steh/bio3afolder/bio3alabmolec
ules.pdf
• http://www.scribd.com/doc/269256/Amino-Acids-Peptides-and-
Proteins
• http://www.steve.gb.com/science/nucleic_acids.html
• http://www.uni-
regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didakti
k/Keusch/p31_d_rib-e.htm