Dna isolation 2011 ec

University of Gondar
Institute of Biotechnology
Techniques in Biotechnology (Biot.602)
Lecture 3
DNA purification/Isolation

 What is DNA (Deoxyribonucleic Acid) ???
Where is DNA?
What are the obstacles to get the DNA?
How to overcome these obstacles?
2Tadele T, April 2010 E.C

 DNA is a nucleic acid that is composed of two
complementary nucleotide polymers
Tadele T, April 2010 E.C 3

Watson & Crick Model of DNA structure
 Purine opposite to a pyrimidine
 A purine always links with a pyrimidine base & held
together by H-bonds
Guanine is paired with cytosine by three H-bonds
Adenine is paired with thymine by two H-bonds
 The linking of the two complementary strands is called
hybridization
 Anti-parallel orientation of the two chains
• 5'-------->3' = One side
• 3'<--------5' = Opposite side
 Two sides twist  double helix

Biological properties
 DNA is metabolically stable, not constantly being degraded &
remade as proteins.
 Each strand of the two strands could serve as a template for
new DNA
 The information is within the unique base sequence of the
DNA.
• Denaturation of DNA duplex strands through one of several
conditions:
– Strong hydrogen bond solvents
– Elevated temperatures
– Alkaline pH

 Since the late 1950s & early 1960s,
molecular biologists have tried to:
• Isolate
• Characterize and
• Manipulate the molecular components
of cells (DNA, RNA & Protein)
• However, how can these cellular
macromolecules be isolated?

 DNA is a hardy and one of the largest known
molecule.
 To work with it, we need to extract it from the other
cellular materials.
Oliver Hardy
Get it- Hardy?

 The genetic engineer may need to
prepare different kinds of DNA:
 total cell DNA from
• Human
• Animal
• Plant
• Bacteria
• Fungi
 plasmid DNA
 phage DNA

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Genetic polymorphism (SNP…) Molecular Cloning PCR Sequencing Southern B
White blood
cells
Plant, animal
tissues
Cultivated
cells
Embrional
cells
Forensic
samples
Fossils
Tadele T, April 2010 E.C

10
The isolation method of choice is depend on:-
 The source of DNA (Plant, blood, tissue,
bacterial)
 The final application (PCR, RE, library
construction)
 The type of DNA (genomic vs. Plasmid)
 The time and expense of method
The quantity DNA

Sample age:
 May be fresh or has been stored .
Stored sample can come from:
◦ Archived tissue samples ,
◦ Frozen blood or tissue (biopsy
material) ,
◦ Exhumed bones or tissues &
◦ Ancient human sample.
 Dried blood spots

 Agricultural applications:-
 Production of healthier crops: crops with disease resistance
 More nutritious ( Genetic engineering of crop plants)
Animal breeding
 Health (Medicine):-
◦ Understanding genetic disorders at molecular level
◦ Rapid detection of genetic disorders in a patient
Novel genes to apply to pharmacological research ? ??
 Environmental (Bioremediation):-
Microorganisms removing pollutants of environment
Industrial:- novel enzymes (able to works at extreme)
 Criminology/Paternity testing
◦ DNA fingerprinting to identify individuals.

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Common Key Steps
 Lysis of the cells
 Separation of the nucleic
acids by removal of
contaminants includes
Proteins
RNA
Other
macromolecules
 Concentration of purified
DNA

 Plant and Bacteria contain lipo-polysaccharides that can,
 Interfere with purification
 Cause toxicity problems in downstream applications.
1. Cell Lysis
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 Tissue- Homogenise, chemically or mechanically
 Cell wall rupture
 Bacteria (Gram-Ve) – lysozyme
 Yeast/fungi - zymolase
 Cell membrane rupture
 Detergents - SDS, triton X-100, CTABCTAB(Cetyl
Trimethyl Ammonium Bromide).
 Proteinases - Proteinase K, Pronase E
 Chelators – EDTA

 CTAB can also;
 Precipitate DNA
 It is also popular to remove polysaccharides.
 Enzymes attacking cell surface components are often added to
detergent-based Lysis buffers.
 Lysozyme digests cell wall components of gram-positive
bacteria.

 Samples which is difficult to lysis by using detergents are first
treated by:
Sonication, grinding in liquid nitrogen and other
shredding devices such as,
 rigid spheres
 beads
 and mechanical stress such as,

• Genomic DNA
SDS/Proteinase K
Alkaline method
Silica column methods
Automated methods
• Plasmid DNA
Alkaline/SDS
Silica column methods
• Bacteriophage DNA
Salt precipitation method

 DNA must be separated from proteins and cellular
debris.
Separation Methods
a) Organic extraction
b) Salting out

19
 Traditionally, phenol: chloroform is used to extract DNA.
 1:1 phenol : chloroform Or
 25:24:1 phenol : chloroform : isoamyl alcohol
 Phenol: denatures proteins, precipitates form at interface
between aqueous and organic layer
 Chloroform: increases density of organic layer
 Isoamyl alcohol: prevents foaming
 When phenol is mixed with the cell lysate, two phases form. DNA
partitions to the (upper) aqueous phase, denatured proteins partition
to the (lower) organic phase.

20
(b) Extraction by organic solvent

21
 At high salt concentration, proteins are dehydrated, lose solubility
and precipitate.
Usually sodium chloride, potassium acetate or ammonium acetate
are used.
Cell lysis.
Protein digestion by proteinase enzyme.
Protein precipitation by high salt concentration.
Centrifugation will remove the precipitated proteins.
The supernatant contains the DNA.
DNA is then precipitated by adding ethanol.
The precipitated DNA is resuspended in the desired buffer.

22
To concentrate nucleic acids resuspension in suitable
buffers, solvents;
 Absolute Ethanol
Isopropanol (final concentration of 40–50%) are commonly used in
the presence of salt to precipitate nucleic acids.
 Both are layered on the top of concentrated solution of DNA and fibers of DNA can
be withdrawn with a glass rod.
 Washing of DNA by using 70% ethanol
 Which is used for desalt of DNA b/c most salts are soluble in 70%
ethanol.

23
 Two ways to concentrate the genomic DNA
“spooling” Ethanol precipitation

A. Nuclease
 One of the major concerns of nucleic acid purification is the
ubiquity of nucleases.
 In a minute after cell dies, the isolation of DNA turns into a
race against internal degradation.
 lysis buffers must inactivate nucleases to prevent nuclease
degradation.
Most lysis buffers contain protein-denaturing and enzyme
inhibiting components.

B. Shearing
 Large DNA molecules,
 Genomic DNA
 Bacterial artificial chromosomes
 Hence Avoid;
Vortexing
Repeated pipetting (through low-volume pipette
tips)
Any other form of mechanical stress.

C. Chemical Contaminant
 Materials that interfere with;
 nucleic acid isolation or downstream applications
involving the purified DNA can originate from the sample.
 Plants, molds, and fungi can present a challenge
because of their
 rigid cell wall
presence of polyphenolic compounds
which can react irreversibly with nucleic acids to
create an unusable final product.

 The reagents/buffers of a DNA purification can also
contribute contaminants to the isolated DNA.
 Reagents that;
 lyse
solubilize samples can inhibit some enzymes when present
in trace amounts.
 Solution
 Ethanol precipitation of the DNA and subsequent
ethanol washes eliminate such a contaminant.

 Phenol can also be problematic. Phenol caused
product oxidation may as result damage DNA.
 A mixture of chloroform and phenol gives a maximize yield of
isolated DNA.
Chloroform reduces the amount of the DNA-containing
aqueous layer at the phenol interphase.
Chloroform can also problematic and should be
removed thorough drying.
® Over dried DNA can be difficult to dissolve, so drying should
be stopped shortly after the liquid can no longer be observed.

 The phenol/chloroform reagent widely used in DNA
purification is notoriously hazardous.
In fact, phenol/chloroform is probably the most hazardous
reagent used regularly in molecular biology labs.
 Phenol is a very strong acid that causes severe burns.
 Chloroform is a carcinogen.
 So, phenol/chloroform is a double whammy.
 It is not only dangerous, but expensive when you
consider the cost of hazardous waste disposal.

 The success of DNA purification is dependent on the initial
quality of the sample and its preparation.
 Ideally start with fresh sample.
 Load the appropriate amount of sample.
 Careful homogenization is crucial
Coldness and gentleness
Inactivate nuclease
 Maintain pH of solution
Avoid UV light, and oxidation by free radicals.

 EDTA is often added to chelate divalent cations required for
nuclease activity and to prevent heavy metal oxidative
damage.
 Tris-based buffers will provide a safe pH of 7 to 8 and will
not generate free radicals,
 Free-radical oxidation seems a key player in
breakdown.
ethanol is the best means to control this process.

 Low temperatures are is important for long-term
stability.
 Storage at 4°C is only for short periods (days).
Storage at -21 or -80 °C is important for long period.
 Another approach for intermediate storage is freeze
drying DNA-containing samples intact.

33
Microbial cell pellet

35
Alkaline
conditions
denature
DNA
Neutralize:
genomic DNA
can’t renature
(plasmids
CAN because
they never
fully
separate)

36
Break down
the cell wall
and
membranes
Dissolve
DNA
Overview of DNA Extraction from plant

37
1- Lyse RBCs & WBCs
2- Lyse WBCs nuclei & Denature/digest proteins
3- Separate contaminants (e.g., proteins, heme)
4- Precipitate DNA
5- Resuspend DNA in TE buffer

END
38
Thank youThank you

DNA quantification & purity
determination

40
 Reliable measurement of DNA concentration is important
for many applications
 DNA quantity and quality can be assessed using several
different methods include:
Absorbance by spectrophotometer or Nanophotometer.
Agarose gel electrophoresis .
 Absorbance: is the most common easies to
determine DNA yield and purity.

Quality of DNA using spectrophotometer
• An instrument employed to measure the amount of
light that a sample absorbs.

42
 The rings of the bases (A, C, G, T, U)
are made up of alternating single
and double bonds.
 Such ring structures absorb in
the U.V.
 Each of the four nucleotide
bases has a slightly different
absorption spectrum, and
 The spectrum of DNA is
the average of them.

◦ DNA UV absorbance at 260nm.
◦ protein >> at 280nm.
◦ Carbohydrate >> at 230nm.
◦ Any insoluble light-scattering components……. absorbance at
320 nm.
Note: Nucleic acids absorb light at 260 nm ,the A260 reading should
be between 0.1–1.0. The spectrophotometer is most accurate when
measurements are in the range of 0.1–1.0.
 However, DNA is not the only molecule that can absorb UV-
light at 260nm.
Since RNA also has a great absorbance at 260nm will
contribute to the total measurement at 260nm

 The ratio of the absorbance at 260 nm/280 nm is a
measure of the purity of a DNA; it should be between
1.7 and 2.0.
 If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinase K to remove protein.
 If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
 DNA Purity (A260/A280) = (A260 reading – A320 reading)
/(A280 reading – A320 reading)

 The ratio of the absorbance at 260 nm/320 nm is a measure
of the purity of a DNA sample from organics and/or salts;
it should be about 2.0.
 Low A260/A320 ratio indicates contamination by organics
and/or salts.
 The absorbance reading indicates how much the sample is pure.

Quantification of DNA by spectrophotometry.
 Using TE buffer as the diluent,
 Make an appropriate dilution of your DNA depending on
the size of the cuvettes available (e.g. for 1ml cuvettes,
dilute 10 microliter DNA solution in 990 micro liters of
TE).
 Determine the absorbance of DNA at 260 nm using TE as the
reference solution (i.e. as a blank).

 Using a conversion factor :
◦ one OD at 260 nm is equivalent to
 Multiply the absorbance reading by
 the conversion factor and
 the dilution factor to find the concentration of nucleic
acid.
 Pure DNA Concentration (microg/ml) =
(A260 reading – A320 reading) x dilution factor x 50microg/ml

 Total yield is obtained by multiplying the
DNA concentration by the final total purified
sample volume.
 DNA Yield (microgram/ml) = DNA
Concentration x Total Sample Volume

 Problem. From a small culture, you have purified the DNA of a
recombinant plasmid. Then you have resuspended the DNA in a
volume of 50 µL TE. You dilute 20 µL of the purified DNA sample
into a total volume of 1000 µL distilled water. You measure the
absorbance of this diluted sample at 260 nm and 280 nm and obtain
the following readings.
A260 --- 0 . 5 5 0
A280 - 0 . 3 2 4
a) What is the DNA concentration of the 50 µL plasmid prep?
b) How much total DNA was purified by the plasmid prep
procedure?
c) What is the A260/280 ratio of the purified DNA?

50
 Don’t need dilution
 The volume required for measurement 3-5
microliters
 The concentration given in nanogram
microliters.

51
 Quality of DNA extracted is assessed using
the following simple protocol:
 Mix 3µL of DNA with 12µL of loading
Dye
 Load this mixture into a 1% agarose gel
 Stain with ethidium bromide
 Electrophorese at 70–80 volts, 45–90
minutes.

Checking for Degradation DNA
 Running your sample through an agarose gel is a
common method for examining the extent of DNA
degradation.
 Smearing indicates
 DNA degradation or
 Too much DNA loaded.

Good quality DNA should
migrate as a high molecular
weight band, with little or no
evidence of smearing.

54
Thank you

Dna isolation 2011 ec

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