ISOLATION AND Stephanie L. Chua, M.Sc.

CHARACTERIZATION OF Dept of Biochemistry

AY 2016-2017
DNA & RNA
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 Polymers of nucleic acids
joined by 3,5-
phosphodiester linkages
NUCLEIC ACIDS Three components
Phosphate group
Five-carbon sugar
Nitrogenous base
Deoxyribonucleic acid
Contains the genetic
material
Serves as the template for
RNA transcription
Ribonucleic acid
Carriers of genetic
information for protein
translation
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 L. Guevarra (2017)

Basis of DNA RNA

Comparison
Sugar deoxyribose ribose

Bases Adenine, guanine, Adenine, guanine,

cytosine, and cytosine, and
thymine uracil
Structure Usually double Single stranded
stranded and sometimes
double stranded
(for some virus)

Location Found in the Found in the

nucleus cytoplasm
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PURINES

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PYRIMIDINES

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 cytosine guanine

ribose deoxyribose

N1 of pyrimidine N9 of purine
to C1 of sugar to C1 of sugar

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Two H bonds for A-T
Three H bonds for G-C

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 THE ISOLATION
PROCESS
Considerations for DNA
isolation
Chemical stability
Condition in the cellular
environment
Experimental factors
1) pH
2) Temperature
3) Ionic strength
4) Cellular conditions
5) Mechanical stress on DNA (C) S. CHUA 2017 9
1) pH
hydrogen bonding in DNA stable only from pH 4 to 10

EXPERIMENTAL FACTORS
phosphodiester bonds are stable from pH 3 to 12
N-glycosyl bonds to purine bases are hydrolyzed at pH 3 or
less

2) Temperature
DNA winds in the range of 80C-90C
Phosphodiester linkages and N-glycosyl bonds are stable
up to 100C

3) Ionic strength
DNA is most stable and soluble in salt solutions
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4) Cellular conditions

EXPERIMENTAL FACTORS
Several enzymes inside the cell degrade DNA, e.g.
deoxyribonucleases
Native DNA exists as DNA-protein complexes

5) Mechanical stress on DNA

Grinding, shaking, stirring, and other disruptive
procedures may cause shearing of DNA
Does not cause damage on the secondary structure

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 THE ISOLATION
PROCESS DNA
FROM ONION

Three major steps:

1. Homogenization
2. Deproteinization
3. Precipitation

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 HOMOGENIZATION

disruption of cell membrane and

release of DNA into a medium that it is
soluble and protected from degradation
made use of a homogenizing
solution and heated to 60C (softens
the phospholipid membrane of the cell
and denatures DNAse)

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THE HOMOGENIZING SOLUTION
Sodium chloride
Most effective in solubilizing and stabilizing DNA
Provides isotonic conditions; separates nucleic acid from other components
Removes histones

Ethylenediaminetetraacetic acid (EDTA)

Binds divalent metal atoms that form salts with the phosphate groups
Inhibits deoxyribonucleases that requires Mg2+ or Mn2+

Sodium citrate
Chelating agent for DNA extraction
Very effective binder for Mn2+ (C) S. CHUA 2017 14
 DEPROTEINIZATION

disruption of protein-DNA complexes

Sodium dodecyl sulfate (SDS)

Anionic detergent that binds to
proteins
Denatures deoxyribonucleases
Papain
Protease to deproteinate DNA

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 PRECIPITATION

ice cold 95% ethanol renders

DNA insoluble in aqueous
solutions
spooling is used to obtain
sticky DNA

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 THE ISOLATION
PROCESS RNA
FROM YEAST

ENZYME
SOLVENT INACTIVATION & ETHANOL
EXTRACTION PROTEIN PRECIPITATION
DENATURATION

1% NaOH soln
Heating at 60 0C 95% Ethanol
Glacial acetic acid with conc. HCl

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PURITY ASSESSMENT BY UV
SPECTROSCOPY
o Absorption of DNA in UV region is primarily due to the
presence of the aromatic bases: A, G, C, T
o Absorbance at 280 nm due to proteins
o Absorbance at 260 nm due to DNA
o A260/A280 : relative measure of DNA to protein content
oTypical value is 1.8 for DNA; 1.9 for RNA
o decreased value indicates high protein contamination

o Hyperchromic shift (increased Abs) is seen because the two

DNA strands are separated

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HYDROLYSIS OF DNA & RNA
1M HCl

Acid hydrolysis
Using 60% HClO4 causes more
1M depurinization than
KOH depyrimidation
DNA cannot be hydrolyzed by
bases because it lacks the 2
hydroxyl group to form the
necessary 2,3 cyclic
monophosphate

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HYDROLYSIS OF DNA & RNA
Alkaline hydrolysis
RNA phosphodiester
bond is unstable
because the OH group
may attack the
electrophilic P to form a
cyclic structure

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 QUALITATIVE
TESTS

Recap of nucleic acid

components:
1) sugar backbone
2) phosphate group
3) nitrogenous bases

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 TEST FOR DEOXYRIBOSE
Dische reaction
Reagents: diphenylamine, conc. H2SO4
Positive result: blue complex
Principle: dehydration and condensation

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 TEST FOR PHOSPHATE
Reagents: concentrated nitric and sulfuric acids, 25%
ammonium molybdate
Positive result: yellow crystals

PO43- + 3NH4+ + 12 MoO42- +

24H+ (NH4)3PO412MoO3 +
12 H2O

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 TEST FOR PURINES
Murexide test
Reagents: concentrated nitric acid, 10% potassium hydroxide
Result: purple red residue of potassium purpurate or red
murexide

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 TEST FOR PYRIMIDINES
Wheeler-Johnson test
Reagents: bromine water and barium hydroxide
Result: purple residue (barium salt of dialuric acid)

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