DNA &RNA
• There are two types of nucleic acids:
ribonucleic acid (RNA) and deoxyribonucleic
acid (DNA).
• Each DNA molecule is very long and usually
consists of hundreds to thousands of
genes.
Structures of nucleic acids (DNA & RNA)
3o 5 Bases
o P o
o DNA Adenine
Base nucleotide (A)
5 CH2
Phosphate
group
o Purine
4 H H 1
H
H
Guanine
3 2 H (G)
o Deoxyribose
o P o
o
Base Cytosine
CH2 o (C) Pyrimidine
H H
H
3 H Thymine
5 3 H
(T)
Sugar-phosphate backbone
Nucleotides
Nucleoside Phosphate
Pentose sugar Nitrogenous base
Ribose Deoxyribose Purines pyrimidines
(RNA) (DNA)
uracil cytosine Thymine
Adenine Guanine
DNA
• Deoxyribonucleic acid
(DNA) is a nucleic acid that
contains the genetic
instructions used in the
development and
functioning of all known
living organisms and some
viruses.
• The main role of DNA
molecules is the long-term
storage of information.
DNA, Deoxyribose Nucleic Acid or
Deoxyribonucleic Acid
DNA determines the characteristics of all living
organisms.
DNA is composed of a four-letter nucleotide/molecule
alphabet referred to as A, T, C, and G.
The order of the alphabet (nucleotides) determines the
characteristics of the living organism, much like the order
of letters in our alphabet determines the words.
Each cell in the human body contains >3 BILLION letters.
DNA is the Blueprint of life
The only difference between living organisms is the
amount and order of the DNA sequence.
Why Extract DNA
The process of extracting DNA from a cell is the first
step for many laboratory procedures in application
DNA.
Good DNA
A good DNA extraction
Pure, suitable quantity and clean of contamination
Is easier to amplify and remain stable for long
time storage.
even optimal (later when PCR is routine).
From what DNA Extraction?
Animals and plants
Blood
Tissues
Body fluids( semen, saliva, etc.)
Bone
Bacteria and Viruses.
All living cell, secretions of living cells, or even dead or
dry parts of living cells (ancient ).
General DNA Extraction pathway
Homogenization of sample
Lysis buffer to lyse cellular and nuclear membranes
Purification from other components
Precipitation of DNA
Washing DNA
DNA Dissolving
Where is the nucleic acid
DNA is packed with proteins to form strand of
chromatin
Disruption of the cell membrane &
nuclear membrane
liberation of the DNA
At this point the DNA must be
protected from enzymes that will
degrade it.
• The DNA in solution
• Thus is the
then first
purified by of DNA extraction uses buffers
stages
• The basicallprinciple
removing otherofcomponents
the (mgcl) nucleicwhich
acidparticularly
extraction consists in releasing the DNA present in
contain inhibitors of nuclease
the inhibitors.
a given matrix by lysis of remaining cell structures.
activity.
• Finally precipitation of the DNA.
Destruction
Cell lysis of cells
Mechanical or Physical Lysis Methods include:
• mechanical agitation
• pressure
• sonication
• nitrogen bomb or nitrogen burst lysis method
• ultrasound with small probes
• Using different forces
• Mechanical, Physical or Biochemical
• N.B: all forces are needed but should be controlled &
standardized during the extraction process
Lysis
1- NaCl so that DNA is stabilised.
2- EDTA which chelates Mg++
and is a co-factor of DNAse .
3- Anionic detergent SDS which disrupts the lipid
layers, helps to dissolve membranes & binds positive
charges of chromosomal proteins (histones) to release
the DNA into the solution.
4-Include a protease (proteinase K) to digest the
proteins
Getting rid of the protein
Organic solvent extraction
using equal volume TE-sat. phenol: chloroform
(24:1) (protein at the interface after centrifugation).
followed by extraction with chloroform:iso-
amylalcohol to remove phenol
OR
Salt-out proteins by precipitation with NaCl or Na-
acetate (protein pelleted after centrifugation.)
Purification of DNA
Precipitation of DNA
Absolute ethanol (Cold) (two volumes).
Isopropanol (Cold) (one volume).
Ethanol and isopropanol
Finally, the ethanol is used to precipitate the DNA.
In water, DNA is soluble. When it is in ethanol, it uncoils and
precipitates leaving behind the other cell components that are not
soluble in ethanol.
• As DNA is completely soluble in water, but not in alcohol, like
isopropanol, when it is added, its engaged more and more water
molecule to interact, as a result, less water molecules are available to
dissolve DNA, and DNA starts ppt out.
• Centrifuging will cause the precipitate to form a pellet
which can be decanted from the unwanted supernatant.
DNA pellet Washed to remove excess salt in 70%
EtOH and air-dry.
Resuspend in sterile distilled water or TE, pH7.4.
Store at 4C or frozen at -20C long term.
Reagents
Reticulocyte saline: 5 mM KCl, 0.13 M NaCl, 7.4 mM MgCl2
10x Lysis mix: 0.77 M NH4Cl, 46 mM KHCO3. Dilute to 2x, to
make red blood cell lysing mix.
WBC Lysing solution: 100 mM NaCl, 25 mM EDTA
10% SDS [Sodium dodecyl sulphate] (make from 20% stock
solution)
10 mg/ml proteinase K
Phenol (saturated with 0.1 M Tris)
Chloroform
7.5 M Ammonium acetate
100% Ethanol (store in ‘flammables cabinet’ with lock)
70% Ethanol
• acetate solution
'dehydrate' the DNA meaning that the DNA is no longer water soluble,
and will therefore precipitate in a watery solution.
Quality of DNA
The product of your DNA extraction will be used in
subsequent experiments.
Poor quality DNA will NOT perform well in PCR or
restrictive digests.
You need to be able to assess the quality of your DNA
extraction.
Check DNA quantity and quality
1. Gel electrophoresis
-Note that genomic DNA
has a very high bp, so one
would expect a
-band at the top of the gel
(near the well) if it was
extracted correctly.
Spectrophotometric analysis of DNA
2. Spectrophotometer
The purity :- OD 260:
OD 280 ratio and must
be between 1.6
-Decreased 260:280 ratio
means the contaminating
protein is present.
-Re-purify sample
Commercial Kits
