KEY STEPS:
Isolation &
characterization
of dna
objectives
• Homogenizing solution:
1. Distinguish different components of DNA
based on qualitative tests
2. Evaluate the quality and concentration of
DNA based on absorbance readings
3. Propose a method to isolate DNA from
biological samples
A. Isolation of dna from onion
- Onions are commonly used because it
contains a lot of DNA
• Blending helps in homogenization (for
• Deproteinization – a protease can be added to
• Precipitation – separation of DNA
*DNA is highly concentrated in the nucleus region
*The cell wall and cell membrane must be
disrupted to access the DNA
- Homogenization (unifying the components
of your solution; one phase) / Cell lysis
- Deproteinization
- Precipitation (separation of DNA from
solution)
- Sodium dodecyl sulfate: detergent used to
lyse cell wall, cell membrane, & nuclear
membrane
- NaCl: stabilizes the negatively charged
DNA (phosphate group)
*when clumped together the surface area
of the molecule; hence, precipitation
- Sodium Citrate: buffer (maintain the
negative charge of DNA)
- EDTA: chelates metal cofactors of
DNAses/nucleases (preserve the integrity
of the DNA molecule)
uniformity)
the homogenized solution (to remove proteins;
increases purity of DNA isolate) ex: papain
(hydrolyze nucleases)
-95% cold ethanol (DNA is insoluble at 65%
EtOH and above)
*water has a higher dielectric constant
compared to EtOH; when high, the interaction of
the (-) charge of the PO4 & (+) charge of Na
atoms will be low
*increase the concentration of EtOH for a
greater interaction between PO4 & Na
*EtOH reacts with water and minimizing the
capability of water to solvate the DNA molecule;
hence, the DNA will only interact with each other
for precipitation
DNA Extraction Kits
- Follows the same principle, but the
protocol might vary depending on the
sample

- DNA purity can be assessed by taking the

ratio of absorbances at 260 and 280 nm
(A260/A280)

B. determination of dna > A260 = nitrogenous bases

A280 = aromatic amino acids
concentration & purity
>

DNA Nomograph
How to use:
1. Plot the absorbances at A260 and A280 at
its respective lines (3rd and 2nd lines in the
- Due to absorbance of the aromatic rings nomograph)
of the nitrogenous bases 2. Connect both points and extend the line
- Strongest at 260 nm towards the outer lines to determine the
A260 ∝ [DNA] protein and nucleic acid concentration
*The aromatic rings nitrogenous bases
have the maximum absorbance at 260 nm
- The concentration of a pure DNA sample
can be estimated by:

Conc (𝜇g/mL) = (A260 reading) x dilution factor x 50 𝜇g/mL

*50 𝜇g/mL is a constant which is the molar

absorptivity of DNA
 Positive result Blue-dark blue solution
C. acid hydrolysis of dna

- Hydrogen bonds between complementary

bases were cleaved
*DNA would melt; strands would separate
- Depurination would occur in mild acidic
conditions
*purine nitrogenous base would be cleaved
from the nucleotide/nucleoside
- A nucleotide undergoes complete hydrolysis *Intensity of blue color is directly proportional to
when subjected to acid hydrolysis the concentration of DNA (deoxyribose)
> Inorganic phosphate
> 2-deoxyribose
b. Test for phosphate (Ammonium
> Nitrogenous bases
molybdate test)
-Differentiates nucleotide from nucleoside
Reagent Conc HNO3,
(NH4)2MoO4, (heat)
Principle Complexation
Detects Inorganic phosphate
Positive result Yellow ppt
d. chemical characterization
of dna

DNA HYDROLYSATE + Test reagent/procedure

a. Test for Deoxyribose

(Diphenylamine/Dische Reaction)
Reagent Diphenylamine (acidic)
Principle Condensation
Detects Deoxyribose
c. Test for purines (Murexide test)
Reagent Conc HNO3, KOH, or
NH4OH
Principle Condensation
Detects Purines
Positive result Yellow to reddish ppt
*any purine

d. Test for pyrimidines (Wheeler-Johnson

test)
Reagent Ba(OH)2, bromine
water
Principle Bromination, then
hydroxylation
Detects Pyrimidines (except
thymine)
Positive result Yellow then purple
solution
*followed by rearrangement