1. Techniques of DNA Extraction, Purification
and Quantification
Submitted to:
Dr. Kapil Tiwari
Assistant Research Scientist,
Bio-Science Research Center,
S.D.A.U., S. K. Nagar
Name : Bhumi A. Gameti
M.Sc. Agri. GPB 3rd sem
Reg. No.: 04-AGRMA-01998-2019
GP 509 - Biotechnology for Crop
Improvement
2. Introduction
DNA (Deoxyribo Nucleic Acid) is a long stringy molecule that can be extracted from any
biological material such as living or conserved tissues, cells and virus particles.
The first isolation of DNA was done in 1869 by Friedrich Miescher.
The extraction of DNA is pivotal to biotechnology. It is the starting point for numerous
applications, ranging from fundamental research to routine diagnostic and therapeutic decision-
making. Extraction and purification are also essential to determining the unique characteristics
of DNA, including its size, shape and function.
The approach to preparation of DNA from plants is determined by the species, the type of
tissue or sample available and the analysis required on the DNA.
4. Uses of isolated genomic DNA
• Preparation of genomic libraries
• PCR template
• Cloning
• Gene/DNA sequencing
• Analysis of genomic organization
• Study gene structure
• DNA fingerprinting
• Analysis of genome composition
• Detection of abnormalities / mutations
5. Extraction of DNA from plant material
Components of the process may
include isolation of specific tissue,
grinding (or other mechanical
disruption), extraction into solution,
solvent purification and
precipitation.
Isolate specific
tissues
Removal of
contaminants
Removal of RNA
contamination
Precipitation
6. Components of DNA extraction solutions
Buffer: Buffers are used to control the pH of the extraction solution.
Salts: Salts may influence the solubility of DNA and other molecules in the extraction.
Chelating agents: Chelating agents such as EDTA bind metal ions in the extraction solution.
Detergents: Detergents help to disrupt tissues. Many different detergents have been employed
in plant DNA extraction protocols.
Phenolic binding agents: PVP is often included to protect against phenolics (Kim et al.,
1997). Citric acid has been added to extracts to prevent the formation of polyphenolics (Singh
et al., 1998).
Other: Activated charcoal has been used to bind contaminants in the isolation of DNA from
coffee, rubber, cassava and banana (Vroh-Bi et al., 1996).
Enzymes: The most common enzyme use in DNA isolation procedures is ribonuclease to
remove contaminating RNA.
Solvents: Water is the solvent used in almost all protocols.
7. Cell Lysis or Cell distruption :
Cell disruption is a method or process for releasing
biological molecules from inside a cell .
Cell Lysis
Physical
Vortexing
Sonication
Manual Grinding
Chemical
Treat with lysozyme,
EDTA, SDS
8. Purification of DNA
• A variety of methods are available for DNA
purification. Generally it is better to devise a method
to isolate DNA of the required purity directly from
the plant sample. Protocols resulting in the isolation
of DNA with specific undesirable impurities should
be avoided. In some cases it may be necessary to
purify DNA in an extract for specific applications.
9. CTAB Method
• The cetyltrimethylammonium bromide (CTAB)
protocol (developed by Murray and Thompson in
1980) is appropriate for the extraction and
purification of DNA from plants.
• CTAB is cationic detergent that has the property of
precipiting nucleic acid and acidic polysaccharides
from solutions of low ionic strength.
11. Phenol–chloroform extraction
A phenol-chloroform extraction is a liquid-liquid extraction.
Based on the differential solubilities of the individual molecules
In 1953, Grassmann & Defner described the efficacy of phenol at
extracting proteins from aqueous solution . Utilizing this find,
Kirby demonstrated the use of phenol to separate nucleic acids from
proteins in 1956 .
Best methods of DNA extraction.
The method is also called as a phenol-chloroform and isoamyl
alcohol, PCI method of DNA extraction.
12. Cont...
Aqueous samples are mixed with equal
volumes of a phenol:chloroform mixture.
After centrifugation, two distinct phases
are formed.
The phenol:chloroform mixture is
immiscible with water.
The aqueous phase is on top because it is
less dense than the organic phase
(phenol:chloroform).
The proteins and hydrophobic lipids will
partition into the lower organic phase
while the nucleic acids (as well as other
contaminants such as salts, sugars, etc.)
remain in the upper aqueous phase.
13. PROTEINASE K
• The proteinase K DNA extraction method facilitates high DNA yield.
• If not maintained well in a cold chain, the proteinase K cannot be utilized
for a longer period of time.
• The lower stability of the enzyme is another major issue in this method.
• This method is a combination of a salt method as well as enzymatic
method. Here the extraction buffer is used before going further on
enzymatic digestion.
• The extraction buffer composition may vary from lab to lab, however, the
major components are Tris, EDTA, NaCl, sodium lauryl and SDS.
14. Cont...
The sample is incubated with proteinase K for 2 hours. This will
digest all the protein present inside the sample.
Immediately after the proteinase K digestion, the sample is
precipitated by chilled alcohol.
By centrifuging sample, all other cell debris are removed.
Finally, the DNA pellet is dissolved in TE buffer.
This method of DNA extraction is rapid and easy. Even the yield is very high. However,
the quality of DNA is a major concern for this method.
15. Salting out
• This method is used to isolate the DNA using the different optimal
concentrations of various salts like Tris-HCl, KCl, MgCl2, NaCl used as the
buffer.
• As a first step in separating the proteins from the DNA, the high concentrations
of NaCl salt is added. The Na+ ions neutralize the negative charge on the DNA,
whereas the Cl- ions neutralize the positive charges on the proteins.
• With their charges neutralized, the proteins and DNA no longer form strong
ionic bonds to one another and therefore can be easily separated.
• The requirement of deproteinizing cell digests with hazardous organic solvents
like phenol, chloroform and isoamylalcohol. This is achieved by salting out the
cellular proteins by dehydration and precipitation with a saturated sodium
chloride solution.
16. Silica adsorption method
DNA separation that is based on DNA molecules binding
to silica surfaces in the presence of certain salts and under
certain pH conditions
The main advantage of silica gel-based DNA extraction is
that it is rapid and gives “PCR ready DNA” for the
downstream applications.
17. There are basic steps:
1.The sample is run through a micro-
channel
2.DNA binds to the channel, and all other
molecules remain in the buffer solution
3.The channel is washed free of impurities
4.An elution buffer removes the DNA
from channel walls, and the DNA is
collected at the end of the channel.
Cont...
18. Magnetic beads
Magnetic particles used for magnetic DNA purification
Magnetic particles made from synthetic polymers, porous glass, or metallic materials
like iron-oxide.
The particles can be coated with functional groups or can be left uncoated. While coated
particles bound with carboxylic acid are the most efficient at binding DNA.
The development of high yield magnetic particles that don't require a coating are
desired because the lack of a coating and functional groups allows a higher surface area
for binding nucleic acid.
Magnetic DNA purification is a clear improvement upon centrifuge-dependent
isolation techniques when semi-automatic or fully automatic systems are considered.
19. Positively charged magnetic beads attract the negatively charged DNA. The DNA is
separated under the magnetic field. DNA extraction buffer is needed in this technique also.
20. FTA Paper
Developed by Burgoyne and Fowler at Flinders
University in Australia in the 1980.
For protecting nucleic acid samples from degradation by
nucleases and other processes.
A sample containing DNA applied to the treated filter
paper for preservation and long term storage.
A marketable advantage of the FTA® technology is that
samples spotted on treated cards may be stored at room
temperature. The chemicals on the FTA cards enhance the
preservation of the DNA and inactivate many dangerous
pathogens that may be found in liquid blood samples or
dried biological stains. Because the cards are small in size
(approximately 3.5” x 5”), they are easily packaged,
shipped, and stored for databasing.
21. NUCLEIC ACID QUANTIFICATION
Quantification of nucleic acids is commonly performed to determine the
average concentrations of DNA or RNA present in a mixture, as well as
their purity.
22. Estimation of the quantity of DNA
• The concentration of DNA in an extract can be measured in
several ways.
• The A260 of the preparation is a good measure of concentration
if the DNA is relatively pure (A260 = 1 for 50 pg ml−1 DNA).
• Impure preparations contain other material absorbing in the
UV range.
• Gel electrophoresis on agarose allows estimation of both
quality and quantity. Fluorometic methods for DNA estimation
are also available. These are likely to be more accurate than
UV-based methods, especially for impure samples.
23. Estimation of the quality of DNA
• The purity of DNA can be estimated from the UV
absorbance (A260/A280 = 1.8 for pure DNA).
• Electrophoresis can be used to assess the size of DNA
molecules in extracts. Digestion with restriction
enzymes is often used to test for DNA purity and
suitability for use in recombinant DNA protocols.
24. Agarose Gel Electrophoresis
This method of quantification is based on the ethidium bromide fluorescent staining
of DNA. Ethidium bromide is a fluorescent dye, which intercalates between the
stacked bases. The fluorescent yield of the dye : DNA complex is much greater
than the unbound dye.
UV irradiation at 254nm is absorbed by the DNA and transmitted to the dye and
the bound dye itself absorbs radiation at 302nm and 366nm. This energy is
retransmitted at 590nm.
The quantity of DNA can be estimated by comparing the fluorescent yield of the
samples with a series of standards.
This provides a very rapid and sensitive means of estimating the nucleic acid
concentration. A large number of samples with as little as 5ng of DNA can be
quantified. Besides quantification, it also allows provides the advantage of
analyzing the quality of the DNA
25. UV spectroscopy
Analysis of UV absorption by the nucleotides provides a simple and accurate estimation of
the concentration of nucleic acids in a sample. Purines and pyrmidines in nucleic acid show
absorption maxima around 260nm (eg., dATP: 259nm; dCTP: 272nm; dTTP: 247nm)
The ratio of OD260/OD280 should be determined to assess the purity of the sample.
The amount of DNA can be quantified using the formula:
DNA concentration (µg/ml) = OD260 x 100 (dilution factor) x 50 µg/ml
1000
Inferences:
A ratio between 1.8-2.0 denotes that the absorption in the UV range is due to nucleic acids.
A ratio lower than 1.8 indicates the presence of proteins and/or other UV absorbers.
A ratio higher than 2.0 indicates that the samples may be contaminated with chloroform or
phenol. In either case (<1.8 or >2.0) it is advisable to re-precipitate the DNA.
26. DNA quantification using NanoDrop
The Thermo Scientific NanoDrop™ 1000 Spectrophotometer measures 1 ul
samples with high accuracy and reproducibility.
The NanoDrop 1000 Spectrophotometer has the capability to measure
highly concentrated samples without dilution (50X higher concentration
than the samples measured by a standard cuvette spectrophotometer).
