PHA6112LAB: PHARMACEUTICAL BIOCHEMISTRY

EXPERIMENT 4: NUCLEIC ACIDS Parts of a Nucleotide

Nucleic Acids 1. Nitrogen Heterocyclic Base

− Pyrimidines - Cytosine (C), Uracil (U) and
̶ biomolecules that store information for
Thymine (T)
cellular growth and reproduction
− Purines - Adenine (A) and Guanine (G)
̶ they are responsible for the storage and
2. Pentose Sugar
passage of genetic information needed for the
− Ribose
production of proteins in every cell and tissue
− Deoxyribose
of an organism
3. Phosphate Residue
̶ DNA - stores the information
− Monophosphate
̶ RNA - synthesizes the proteins
− Diphosphate
̶ the proteins produced in the cell are needed to
− Triphosphate
make other proteins, carbohydrates, lipids,
and nucleic acids

Types of Nucleic Acid

1. Deoxyribonucleic Acid (DNA)

2. Ribonucleic Acid (RNA)
̶ these are polymers consisting of long chains of
monomers called nucleotides

Difference between DNA and RNA

DNA and RNA have distinct characteristics that affect their isolation and testing. Table 1 presents the differences
between DNA and RNA based on parameters such as distribution in cell, nucleotide component, nitrogenous base
interactions, structure and synthesis.

Table 1. Difference between DNA and RNA

PARAMETER DNA RNA

Distribution in Cells Prokaryote Exists in single molecule in a Found in the cytoplasm
circular double helical form in
the cytoplasm
Eukaryote Exists in linear double helical - Found in cytoplasm in a complex with non-histone
form in the nucleus complexed proteins in ribosomes
with histone, mitochondrion - Also found in nucleolus where ribosomes are
and chloroplast synthesized
-There are 3 classes:
-rRNA
-mRNA
-tRNA
Nucleotide Component Sugar Deoxyribose Ribose
Pyrimidine Bases AGC + Thymine AGC + Uracil

Interaction between Bases Intermolecular H-bonding Intramolecular H-bonding

Polynucleotide Chain 2 polydeoxyribonucleotide 1 polyribonucleotide chain (single strand)
chain (double strand)
Synthesis Synthesized as the cell divides Synthesized as the need arises
-short-lived
Properties of Nucleic Acids RNA

̶ because it is single stranded, it is less viscous

I. Structure
than DNA solutions
DNA
III. Solubility
̶ consist of a double helix due to the H-bonds
between complementary bases, A-T and G-C SOLVENT SOLUBILITY
Salt Solutions with soluble
Denaturation High Concentration
Weak Alkali soluble
̶ the unwinding of the DNA double helix - e.g. NH3
resulting from a break in the H-bonds Cold Water sparingly soluble
between bases GC and AT Alcohol insoluble
Salt Solutions with insoluble
Renaturation or Annealing
Low Concentration
̶ the rewinding of the separated DNA strands,
restoration of H-bond between bases GC and
IV. UV Absorption
AT
̶ nitrogenous bases are absorbed by UV light
strongly at 260 nm

DNA

̶ unwinding (denaturation) of DNA causes

disruption of H-bonding between strands
which exposes the N bases resulting into a
“hyperchromic effect”
̶ “Hyperchromic Effect”
RNA
− increased absorbance at 260 nm
̶ single stranded consisting of nitrogenous ̶ rewinding (renaturation or annealing) causes
bases C, U, G and A “hypochromic effect”
̶ “Hypochromic Effect”
II. Consistency − decreased absorbance at 260 nm

DNA RNA

̶ solutions are viscous at pH 7.0 & 25°C due to ̶ generally have higher absorption than DNA
rigidity of the double helix and high because it is single stranded
length/diameter ratio
Basis for Evaluating the Purity of Nucleic Acid
̶ DNA becomes less viscous at high
Extracts (Purity of Nucleic Acids)
temperatures
̶ Transitory Temperature (Tm) or Melting ̶ A260/A280
Point − used as relative measure of NA/protein
− the temperature at which 50% of the content of a DNA sample
double helix is unwound − Proteins (i.e. with WYF) - absorb strongly
− ↑ higher Tm = ↑ more GC pairs at 280 nm
− ↓ lower Tm = ↑ more AT pairs − Good Quality DNA Sample - ranges from
̶ when T and pH is returned to the optimum 1.8-2.0
range: − Pure Isolated DNA - 1.8
− DNA anneals and viscous consistency is
restored
 − if absorbance is <1.8 = ↑ increased − ↑ more dense DNA = migrate downward
contamination by protein ↓
− if absorbance is >1.8 = ↑ increased − ↓ less dense DNA = migrate upwards ↑
contamination of RNA or denaturation of forming bands
DNA
̶ A260/A230
− used as relative measure of NA/X (or
other contaminants e.g. polysaccharides,
phenols or salts)
− these contaminants absorb strongly at
230 nm
− Ideal Value - 2.0

Optical Density Monogram

̶ used to estimate the amount of protein

contaminants and isolated NA
Experimental Factors that Affect Nucleic Acids

1. pH

H-bonding stable between

between Strands pH 4-10
Phosphodiester stable between
Linkages in the pH 3-12
DNA backbone
β-N-glycosidic hydrolyzed at
Bonds to Purine pH ≤ 3 or less
Bases
DNA hydrolyzed at
pH < less than 3 or >
greater than 12

Steps
2. Temperature
1. Using a ruler, connect the two points of the
OD 280 nm and 260 nm. DNA unwinds at 80-90°C
2. Draw a line across the monograph (running Phoshodiester and β- stable up to 100°C
from the “protein” concentration to the N-glycosidic Bonds
“nucleic acid” concentration) with the ODs as
the two points connecting the line
3. Ionic Strength and Solubility
3. e.g. OD 280 nm is 0.5; OD 260 is 0.6
Approx conc. of protein contaminants is SOLVENT OF DNA SOLUBILITY
0.32 mg/mL (& STABILITY)
Approx conc. of nucleic acid is 16 mcg/mL in Salt Solutions with most stable and
High Concentration soluble
V. Density (DNA) in Salt Solutions with insoluble
̶ measured by CsCl density ultracentrifugation Low Concentration
̶ Cesium Chloride (CsCl) ̶ Salt Solutions less than 0.1 M - weakens H-
− forms a density gradient with the most bonding
dense solutions at the bottom
− DNA concentrates on CsCl area because it
has the same density as CsCl
 4. Mechanical Stress
− e.g. grinding, shaking, stirring, swuirting
through narrow orifices and others
− DNAs
− are cleaved (shearing or scission)
− does not cause damage to secondary
structure but it reduces the lengths of
the molecules
− RNAs
− are easily damaged by shearing
5. Cellular Conditions
− Nucleases (DNase & RNase)
− catalyze hydrolysis of 3’-5’
phosphodiester linkage making up the
NA backbone
− Nucleases Present in Human Fingertips
− can cause spurious degradation of
nucleic acids during purification
− glass, rubber or plastic tools and
containers - used to avoid degradation
of DNA
6. Preservation
− DNA
− Purified DNA - best kept in solution
− Secondary Structure - kept at 0.1M
acetate buffer
− Addition of 0.001M Sodium Azide -
inhibits growth of microorganisms
− Suitable Storage Temperature (T) - 4°C
(frozen state)
− RNA
− RNAs of LMW - best kept as dried
powder
− RNAs of HMW - best kept as slurry
under 75% aq. alcohol containing 2%
sodium azide at 4°C
General Principles in the Isolation of Nucleic Acids
1. Homogenization
− disruption of cell membrane and
organelle membranes releases the
nucleoprotein (DNA-histone complex)
into a medium in which it is soluble and
protected from degradation
− Denaturation of Enzymes - must be done
to inactivate nucleases
MECHANICAL mincing, grinding, sonication,
etc.
CHEMICAL detergents & chaotropic agents
2. Dissociation and Denaturation of
Nucleoprotein
− separates the protein and releases the NA
− NA is then precipitated out
− it is removed by spooling after the NA has
been precipitated out
3. Purification of Nucleic Acid (NA)
− it is difficult to isolate it in an intact and
undamaged form because of the large
and fragile nature of nucleic acids
− Isolation - should be conducted where
drastic changes in conditions (see
experimental factors affecting NA) are
avoided or minimized
Choice of Sample for DNA
̶ tissues containing cells with high nuclear
volume/ cytoplasmic volume ratio
MICROBIAL DNA -Bacillus subtilis or
Eschericia coli
PLANT DNA -meristematic region of
any plant & yellow
onion
ANIMAL DNA -spleen, liver, thymus
and pancreas
-brain and muscle
tissues - low
concentration
I. Isolation of Microbial DNA
̶ target for extraction is the disruption of
bacterial cell wall and the inactivation of
enzymes
Materials used for Isolation
̶ Lysozyme
− used to cause lysis of bacterial cells by
hydrolyzing the peptidoglycan present in
cell walls
− this disrupts the bacterial cell wall to let
the DNA be in solution
 ̶ EDTA (ethylenediaminetetraacetic acid)
− used as chelating agent for divalent cations
(Mg++) for the metals present in DNAses
− this inactivates the enzyme
II. Isolation of Plant DNA (Meristematic Region)
Processes used for Isolation
̶ Heating at 60°C
− dissolves nucleic acids
̶ Cooling in Ice Bath
− retards nuclease activity

̶ one of the most difficult DNAs to isolate IV. Isolation of Animal DNA
because of the:
1. Structure of the Plant Cell ̶ Lysing Buffer
− hard, rough, solid cell wall because of − 5M Sodium Chloride
peptidoglycan, pectin, cellulose and − osmosis in the cell
chitin − Tris HCl
2. Components of the Plant Cell − buffer
− secondary metabolites − 0.5M EDTA
− e.g. polysaccharides, phenols, etc. − chelates the metals in the DNases
− 5% SDS
Materials used for Isolation − disrupts cell membrane and nuclear
envelope
̶ CTAB (Cetyl trimethylammonium bromide) ̶ Chloroform
− a cationic detergent used to separate − denatures the proteins and lipids to
polysaccharides during purification of DNA maintain separation of organic and
samples from plants aqueous phases
̶ NaCl ̶ Isoamyl Alcohol
− removes proteins that are conjugated to − prevents foaming
the DNA
− proteins are kept dissolved in the aqueous RNA
portion which prevents alcohol from
precipitating it along with DNA ̶ single-stranded NA found in high
concentrations in tissues with large
III. Isolation of DNA from Onion cytoplasmic volume

Materials used for Isolation Choice of Sample

̶ Homogenizing Solution ̶ ↓ nuclear-cytoplasmic volume ratio because

− 0.15M NaCl RNA concentration is high in the cytoplasm
− precipitates nucleoproteins (salting- ̶ Saccharomyces cerevisiae (yeast)
out) − 4% RNA by weight
− 5% SDS (sodium dodecyl sulfate)
− breaks ionic interaction between Isolation of RNA from Yeast
protein and nucleic acid
̶ Heating with Dilute NaOH
− 0.15M Sodium Citrate
− separates RNA from proteins
− chelates Ca2+ and Mg2+ ions (cofactors)
− extracts RNA and water-soluble proteins
− 1 mM EDTA
− inactivates nucleases (RNase)
− chelates Ca2+ and Mg2+ ions (cofactors)
̶ Glacial Acetic Acid (pH 4-5)
− Papain or Meat Tenderizer 6%
− separates nucleic acids associated with
− denatures protein
proteins and other interfering substances
− Ice-cold 95% EtOH
̶ Ethanol with Concentrated HCl
− precipitates DNA and also RNA
− precipitates RNA
− Ice-cold 100% iPrOH
̶ Alcohol (EtOH) and Ether (organic solvents)
− precipitates DNA only
− removes lipids
 ̶ TE buffer & SSC (Standard Saline Citrate) RNA has a higher absorption at 260 nm than DNA.
− preserves integrity of RNA by maintaining Hence, a larger/higher extinction coefficient. This
the pH of the solution phenomenon is attributed to RNA’s structure (single
-stranded) where nitrogenous bases are exposed as
UV Measurement of Nucleic Acids opposed to DNA (double-helical structure). The
̶ Absorbance conversion factor computed here is for double-
− measured at 260, 280 and 230 stranded DNA. Single-stranded DNA has 33 ug/mL if
− Nucleic Acids (DNA and RNA): λmax - 260 Abs = 1.000 and 30 ug/mL for oligonucleotide
nm solution.
− Presence of aromatic nitrogenous
bases - purines and pyrimidines
− Structural changes resulting from
Table 2. Extinction and Standard Coefficients of
denaturation (i.e. helix unwinding)
Nucleic Acids
− Primary Protein Contaminants: λmax - 280
nm NA Extinction 1 cm
Coefficient Pathlength
Estimation of Standard Coefficient Standard
𝐴 = 𝜀𝑏𝑐 Coefficient
mcg/mL
where: dsDNA 0.020 50
ssDNA 0.025 40
A - absorbance ssRNA 0.027 33

ε - extinction coefficient
Formula for the Concentration of the Isolated
b - pathlength Nucleic Acid
c - concentration of NA 𝑂𝐷260
𝐷𝑁𝐴 = ( ) 𝑥 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 𝑥 𝑠𝑎𝑚𝑝𝑙𝑒 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛
𝑝𝑎𝑡ℎ𝑙𝑒𝑛𝑔𝑡ℎ
For DNA:
Standard Coefficient - obtained from Table 2.
The average ε of DNA = 0.020 (mcg/mL)-1 cm -1
Sample Problem
If A260 = 1.00 and b = 1, then
A solution of DNA (in TE buffer) gave an A260=0.55.
CDNA = 50 mcg/mL The absorbance of the buffer at 260 nm is 0.15. What
is the concentration of DNA in the original solution a)
For RNA: assuming without dilution and b) if the DNA was
diluted to 1:20 prior to measurement of absorbance?
The average ε of RNA = 0.025 (mcg/mL)-1 cm -1
a) CDNA = A260 x 50 mcg/mL x dilution
If A260 = 1.00 and b = 1, then
= (0.55-0.15) x 50 mcg/mL x 1
CRNA = 40 mcg/mL
= 20 mcg/mL or 20 ng/mcL
For accurate readings, the NA sample of interest
should be diluted to give readings between 0.1 to b) CDNA = A260 x 50 mcg/mL x dilution
1.0.
= (0.55-0.15) x 50 mcg/mL x (1/20)

= 1 mcg/mL or 1 ng/mcL
Hydrolysis of Nucleic Acids insoluble thereby optimizing precipitation of
̶ Nucleic acids are hydrolyzed to separate its the product.
components for qualitative tests. ̶ The phosphate ion reacts with ammonium
molybdate in nitric acid to form a yellow
crystalline precipitate of ammonium
phosphomolybdate.
̶ The precipitate is formed slowly from the
solution.
̶ Positive Result - yellow crystalline ppt

Ionic Representation of the Reaction

Test for Sugars

Acid Hydrolysis
̶ Diphenylamine or Dische (1930) Test for
̶ produces brown-black solid Deoxyribose
− Reagent - diphenylamine
Alkaline (Basic) Hydrolysis in conc. H2SO4
− Positive Result - blue complex/compound
̶ not complete for RNA and a mixture of 2’ and
λmax = 595 nm
3’ nucleotides are produced

DNA is not readily hydrolyzed by dilute alkali because

it has no 2’-hydroxyl group and therefore, cannot
form the necessary 2’,3’-cyclic monophosphate.

Enzymatic Hydrolysis

̶ nucleic acids may also be hydrolyzed

enzymatically by nucleases which exhibit
This reaction is given by 2’-deoxypentoses in general
different specificities:
and is not specific for DNA. In DNA, only the
− specificity for the sugar
deoxyribose of the purine nucleotide reacts so that
− specificity for phosphodiester bonds at
the value obtained represents half of the total
the end (exonucleases) or anywhere in a
deoxyribose present.
chain (endonucleases)
− specificity for the secondary structure
̶ Bial’s Orcinol Test for Ribose
− specificity for the base
− Reagent - Orcinol in HCl
Qualitative Tests for Nucleic Acids (yellow solution)
− Positive Result - blue-green
Test for Phosphates (PO4-3) coloration

̶ The mixture is heated to remove excess

sulfuric acid as colorless sulfur trioxide gas.
̶ Concentrated nitric acid is added to provide a
strongly acidic medium in which the product is
This reaction is not absolutely specific for pentoses
since prolonged heating of some hexoses yields
hydroxymethyl furfural which also reacts with orcinol
to give a colored complex (False-positive result). This
test is also not specific for ribose, it is a general test
for pentoses (5-C monosaccharides). It is frequently
employed for the estimation of RNA.

Test for Nitrogenous Bases

̶ Murexide Test for Purines

− Positive Result - red coloration due to the
formation of murexide

̶ Wheeler-Johnson Test for Pyrimidines

− Sx → bromine water →boil to remove
excess → Ba(OH)2 → purple color due to
Ba+2 salt of dialuric acid
− Positive Result - purple coloration