STRUCTURE, FUNCTION AND
EXTRACTION METHODS OF DNA
• Fundaments on
• Genes, DNA, RNA,
Genome and Genetics
Anil kumar Sah
Senior Research Officer
Annapurna Research Center
Principle information
Nucleicmolecule
acids in the cell.
All the genetic codes are carried out on the
nucleic acids.
Nucleic acid is a linear polymer of
nucleotides
Nucleotides are the unit structure of nucleic
acids. Nucleotides
Nucleotides composed of 3 components:
• Nitrogenous base (A, C, G, T or U)
• Pentose sugar
• Phosphate
There are 2 types:
• Purines:
Nitrogenous bases
• Two ring structure
• Adenine (A) and Guanine (G)
• Pyrimidines:
• Single ring structure
• Cytosine (C) and Thymine (T) or Uracil (U).
DNA is a doubleDNA structure
stranded molecule consists of 2
polynucleotide chains running in opposite directions.
Both strands are complementary to each other.
The bases are on the inside of the molecules and the 2
chains are joined together by double H-bond between
A and T and triple H-bond between C and G.
The base pairing is very specific which
DNA structure
make the 2 strands complementary to
each other.
So each strand contain all the required
information for synthesis (replication) of
a new copy to its complementary.
The DNA double strands can denatured if heated
Denaturing
(95ºC) and
or treated with Annealing of DNA
chemicals.
• AT regions denature first (2 H bonds)
• GC regions denature last (3 H bonds)
DNA denaturation is a reversible process, as
denatured strands can re-annealed again if cooled.
This process can be monitored using the
hyperchromicity (melting profile).
Hyperchromicity
It is used (meltingandprofile)
to monitor the DNA denaturation annealing.
It is based on the fact that single stranded (SS) DNA gives
higher absorbtion reading than double stranded (DS) at
wavelength 260º.
Using melting profile we can differentiate between single
stranded and double stranded DNA.
The alphabet ofGenetic
the genetic code
code contains only four
letters (A,T,G,C).
A number of experiments confirmed that the genetic
code is written in 3-letter words, each of which
codes for particular amino acid.
A nucleic acid word (3 nucleotide letters) is referred
to as a codon.
In water, plants, soil and humans.
Forensic Specimen
Parasites
Fungi
Bacteria
Viruses
The success of modern medicine depends on the
Molecular Diagnostics
detection of specific molecules e.g.
Sensitivity means that the test must be able to
detect very small amounts of target even in the
presence of other molecules.
Simplicity
Specificity
Sensitivity
System
A good detection system should have 3 qualities:
Characteristics of a Detection
Specificity: the test yields a positive result for the
target molecule only.
Simplicity: the test must be able to run efficiently and
inexpensively on a routine basis.
Why use a molecular test to diagnose an infectious disease?
• Importa
• Importa
Need an accurate
disease
ous
contagi
of a
spread
ng the
preventi
nt for
nt
treatme
proper
g the
initiatin
nt for
and timely diagnosis
Leading uses for nucleic acid based tests:
Noncultura •Human papilloma virus
ble agents •Hepatitis B virus
Fastidious, slow- •Mycobacterium
growing agents tuberculosis
Highly infectious •Brucella species
agents that are
dangerous to culture •Coccidioidis immitis
Leading uses of nucleic acid based
tests:
In situ detection •Helicobacter pylori
of infectious
•Toxoplasma gondii
agents
Agents present •HIV in antibody negative patients
in low numbers •CMV in transplanted organs
Organisms •Intra-ocular fluid
present in small
volume specimens
•Forensic samples
What are the essential components of a DNA
extraction Procedure?
Remove or Maximize the
Maximize Remove
inhibit quality of
DNA recovery inhibitors
nucleases DNA
How Much DNA Do We Need?
The PCR reactions call for on average 1 ng of DNA (single or double stranded).
Many of the commercially available kits are sensitive below 1 ng of DNA (100-
250 pg).
Plant Cell
Animal Cell
Most Commonly used DNA
Extraction Procedures
•Organic (Phenol-Chloroform)
Extraction
•Non-Organic (Proteinase K
and Salting out)
The method utilized may be sample dependant, technique dependant, or analyst
preference
Extraction Buffer
Detergents
1. Dissolve cellular membranes Chaotropic salts
CTAB
Detergents
2. Inactivation of DNase and Rnase Metal chelators
Reducing agents
3. Assist in the removal of contaminants Salts
CTAB
PVP
Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation
Before After
Supernatant 70% EtOH
Centrifuge Wash Centrifuge
Pellet
Dissolve pellet
(H2O, TE, etc.)
Add alcohol and salt to • Pellet down nucleic acids.
precipitate nucleic acids • Wash pellet with 70% ethanol to remove
from the aqueous fraction residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
Membrane based method
A spin column using a silica-based extraction method is used. This does not
require the use of hazardous chemicals. Nucleic acids are attracted to the silica
bead under high chaotropic salt concentrations. The sample and lysis buffer are
added to a sterile tube.
The lysate is combined with alcohol and placed into
the spin column, which is inserted into a tube. The
removal of proteins and divalent cations is
accomplished using multiple buffer washes and
centrifugation steps. Removal of cations, such as
Mg2+, prevents nucleases from further degrading
the DNA. Pure DNA is eluted from the membrane
into sterile water or TE buffer.
Basic steps for DNA extraction
• Breaking the cells, commonly referred to as cell
disruption or cell lysis, to expose the DNA within.
• This is commonly achieved by grinding, sonicating
or treating the sample with lysis buffer .
• Removing membrane lipids /Polysaccharide by
adding a detergent/lysozyme.
• Removing proteins by adding
a protease/Proteinase K
• Precipitating the DNA with an alcohol
usually ice-cold ethanol or isopropanol.
Nucleic Acid Precipitation
Before After
Supernatant 70% EtOH
Centrifuge Wash Centrifuge
Pellet
Dissolve pellet
(H2O, TE, etc.)
Add alcohol and salt to • Pellet down nucleic acids.
precipitate nucleic acids • Wash pellet with 70% ethanol to remove
from the aqueous fraction residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
• Depending on the material and requirement
the extraction process can be modified with
alteration of chemicals used
• DNA can be stored at 4˚C for extended
periods, however for long term storage, - 20˚
C is preferable.
• Avoid repetitive freeze thawing of DNA, since
this can cause degradation.
DNA Extraction By CTAB Methods
• At least 1 ml of culture suspension/Specimen
was centrifuged at 10,000 rpm for 10 min.
• The supernatant was discarded and the pellet
suspended in 567μl of TE buffer (Tris EDTA, pH
7.4), 30 μl 10% SDS and 3μl proteinase K (20
mg/ml), and mixed.
• Then incubate at 37°C for 1h.
DNA Extraction…….
• After incubation, 100μl of 5 M NaCl and 80μl
of high-salt CTAB buffer (containing 4 M NaCl,
2% CTAB) were added and mixed, followed by
incubation at 65°C for 10 min.
• Then approximate equal volume (0.7–0.8 ml)
of chloroform-isoamylalcohol(24:1) were
added, mixed thoroughly and centrifuged for
4–5 min at 12,000 rpm.
DNA Extraction…….
• The aqueous viscous supernatant was carefully
decanted and transferred to a new tube.
• An equal volume of phenol: chloroform: isoamyl
alcohol (25:24:1) were added, followed by a 5 min
spin at 12,000 rpm.
• The supernatant were separated and then mixed
with 0.6ml of isopropanol to get a precipitate.
DNA Extraction…….
• The precipitate nucleic acids were washed with
75% ethanol, dried and re-suspended in 40μl of
TE buffer.
• Stored at - 20˚ C
Assessing the Quality and Yield
of Nucleic Acids
Checking for DNA
Running nucleic acid sample
genomic through an agarose gel is a
DNA
common method for
examining the extent of DNA
degradation. Good quality
RNA
(degraded)
DNA should migrate as a high
molecular weight band, with
little or no evidence of
smearing.
Nucleic Acid Analysis via UV
Spectrophotometry
DNA Absorption Spectra
By measuring the amount of light absorbed by your sample at specific
wavelengths, it is possible to estimate the concentration of DNA and
RNA. Nucleic acids have an absorption peak of 1 OD at ~260nm.
[dsDNA] ≈ A260 x (50 µg/mL)
[ssDNA] ≈ A260 x (33 µg/mL)
[ssRNA] ≈ A x (40 µg/mL)
Nanodrop, Flourimeter
Thank You