PROPERTIES OF NUCLEIC ACIDS

DNA RNA

Consist of a double helix 
 single-stranded

due to the H-bonds between consisting of nitrogenous bases A, U,
complementary bases, A-T and G-C. 
 G, C

DENATURATION
Structure – the unwinding of the DNA double helix
resulting from a break in the H-bonds
between bases GC and AT.

RENATURATION OR ANNEALING
– the rewinding of the separated DNA
strands, restoration of H-bond between
bases GC and AT

Solutions Because it is single-stranded, 


- viscous at pH 7.0 & 25°C 
 it is less viscous than DNA solutions.
- due to rigidity of the double helix 

& high length/diameter ratio
- less viscous at high temperatures
(80-90°C)

- MELTING POINT or


TRANSITORY TEMPERATURE (Tm)

Consistency
— temperature at which 50% of the double
helix is unwound 


- Higher Tm — more GC pairs; 


Lower Tm — more AT pairs. 


*When T and pH is returned to the optimum

range, the DNA anneals and viscous
consistency is restored.

Soluble in salt solutions with high concentration.

in weak alkali such as NH3.

Solubility Sparingly soluble in cold water

Insoluble in alcohol
in salt solutions with low concentration

Nitrogenous bases are absorbed by UV light strongly at 260 nm

Unwinding (denaturation) of DNA

 Unwinding (denaturation) of DNA
- causes disruption of H-bonding 
 Generally have higher absorption than
DNA because it is single stranded
between strands — exposes the N bases
resulting into a “hyperchromic effect” 



Hyperchromic effect

— increased absorbance at 260 nm

Rewinding (renaturation or annealing)

- causes “hypochromic effect”



Hypochromic effect”

— decreased absorbance at 260 nm
UV Absorption

This is the basis for evaluating the purity of nucleic acid extracts. 

Purity of nucleic acids is expressed as
! A260/A280 – used as relative measure of NA/protein content of a DNA sample. 

Proteins (i.e. with WYF) absorb strongly at 280 nm. 

Good quality DNA sample ranges from 1.8-2.0 

Pure isolated DNA is 1.8. 

Therefore, if absorbance is < 1.8, there is increased contamination by protein; 

if absorbance is >1.8, there is an increased contamination of RNA or denaturation of DNA. 


! A260/A230 - used as relative measure of NA/X or 


other contaminants such as: polysaccharides, phenols or salts. 

These contaminants absorb strongly at 230 nm. Ideal value is 2.0.



Amount of protein contaminants and isolated NA 

— estimated using the Optical Density Monogram

QUALITATIVE TESTS

The mixture is heated — remove excess sulfuric acid 


→ colorless sulfur trioxide gas. 



Concentrated nitric acid — added to provide a 

strongly acidic medium in which the product is insoluble

→ optimizing precipitation of the product



Phosphate ion 

— reacts with ammonium molybdate in nitric acid 

→ yellow crystalline precipitate of 

TEST FOR PHOSPHATES (PO4-3) ammonium phosphomolybdate



*The precipitate is formed slowly from the solution. 



Ionic representation of the reaction
:
PO4-3 + H2SO4 → H3PO4
x’ss H2SO4 + Δ → SO3 ↑
PO4-3 + 3NH4+ + 12MoO4-2 +24H+ →
(NH4)3PO4·12 MoO3↓ + 12 H2O
yellow crystalline ppt
 QUALITATIVE TESTS

(for Deoxyribose) DIPHENYLAMINE OR DISCHE (1930) TEST

Reagent: diphenylamine in conc. H2SO4

Positive Result: blue complex/compound (λmax = 595 nm)
This reaction is given by 2’-deoxypentoses in general and is not specific
for DNA. In DNA, only the deoxyribose of the purine nucleotide reacts so
that the value obtained represents half of the total deoxyribose present.

(for Ribose) BIAL’S ORCINOL TEST

TEST FOR SUGARS
Reagent:
Positive Result: blue-green coloration
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.

(for Purines) MUREXIDE TEST

Result: Red coloration — due to the formation of murexide.

TEST FOR 

NITROGENOUS BASES

(for Pyrimidines) WHEELER-JOHNSON TEST

Result: Purple coloration


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