WRITE EXTENSIVELY ON PROCEDURES OF DNA EXTRACTION
TABLE OF CONTENT
Introduction
1.1 Background Information on DNA Extraction
1.2 Importance of DNA Extraction in Biological Research
Fundamentals of DNA
2.1 Structure of DNA
2.2 Function of DNA in Living Organisms
Purpose and Applications of DNA Extraction
3.1 Research Applications
3.1.1 Genetic Studies
3.1.2 Forensic Analysis
3.2 Medical Applications
3.2.1 Disease Diagnosis
3.2.2 Pharmacogenomics
Basic Principles of DNA Extraction
4.1 Cell Lysis
4.2 Denaturation
4.3 Precipitation
4.4 Purification
Types of DNA Extraction Methods
5.1 Phenol-Chloroform Extraction
5.2 Salting Out
5.3 Silica-Based Extraction
5.4 Commercial DNA Extraction Kits
Selection of Source Material
6.1 Blood
6.2 Tissue Samples
6.3 Buccal Swabs
6.4 Plant Material
Laboratory Procedures for DNA Extraction
7.1 Sample Collection
7.2 Cell Disruption
7.3 DNA Purification
7.4 Quality Control
Challenges and Troubleshooting
8.1 Contamination Issues
8.2 Low DNA Yield
8.3 Inhibitors in Samples
Conclusion
INTRODUCTION
DNA was first isolated by the Swiss physician, Friedrich Miescher, in 1869 while
working in the laboratory of the biochemist Felix Hoppe-Seyler. This he did as part of
a project to determine the chemical composition of cells which he saw as the means to
unravelling the fundamental principles of the life of cells. Initially, he began this
research by using lymphocytes drawn from lymph nodes but was unable to get
sufficient quantities for analysis so switched to using leukocytes, white blood cells,
which he gathered from pus found on fresh surgical bandages collected from a nearby
surgical clinic. In the course of his work on leukocytes he noticed the precipitation of
a substance when acid was added and that this dissolved following the addition of
alkali. Miescher decided to call the new substance 'nuclein by virtue of its presence in
the nuclei of the cell. Upon further analysis, Miescher noted that the chemical
composition of the substance differed from proteins and other known molecules. He
speculated that it played a central role in cells and was involved in the division of
cells. Following this, Miescher developed a method for isolating nuclein from salmon
sperm. Many advances have been made to the methods for extracting and purifying
DNA since Miescher's time. From the 1950s routine laboratory extraction of DNA
became reliant on the use of density gradient centrifuges. Until very recently most
methods for extracting DNA remained complex, labour-intensive and time-
consuming. They also provided only small quantities of DNA. Today there are many
specialised extraction methods. These are generally either solution-based or column-
based. The extraction of DNA has become much easier with the emergence of
commercial kits and the automation of the process. Such changes have both sped up
production and increased the yield of DNA.
The ability to extract DNA is of primary importance to studying the genetic causes of
disease and for the development of diagnostics and drugs. It is also essential for
carrying out forensic science, sequencing genomes, detecting bacteria and viruses in
the environment and for determining paternity.
DNA
Deoxyribonucleic acid is a polymer composed of two polynucleotide chains that coil
around each other to form a double helix. The polymer carries genetic instructions for
the development, functioning, growth and reproduction of all known organisms and
many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins,
lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four
major types of macromolecules that are essential for all known forms of life.
The two DNA strands are known as polynucleotides as they are composed of simpler
monomeric units called nucleotides.[2][3] Each nucleotide is composed of one of four
nitrogen-containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine
[T]), a sugar called deoxyribose, and a phosphate group. The nucleotides are joined to
one another in a chain by covalent bonds (known as the phosphodiester linkage)
between the sugar of one nucleotide and the phosphate of the next, resulting in an
alternating sugar-phosphate backbone. The nitrogenous bases of the two separate
polynucleotide strands are bound together, according to base pairing rules (A with T
and C with G), with hydrogen bonds to make double-stranded DNA. The
complementary nitrogenous bases are divided into two groups, the single-ringed
pyrimidines and the double-ringed purines. In DNA, the pyrimidines are thymine and
cytosine; the purines are adenine and guanine.
DNA is the information molecule. It stores instructions for making other large
molecules, called proteins. These instructions are stored inside each of your cells,
distributed among 46 long structures called chromosomes. These chromosomes are
made up of thousands of shorter segments of DNA, called genes. Each gene stores the
directions for making protein fragments, whole proteins, or multiple specific proteins.
DNA is well-suited to perform this biological function because of its molecular
structure, and because of the development of a series of high performance enzymes
that are fine-tuned to interact with this molecular structure in specific ways. The
match between DNA structure and the activities of these enzymes is so effective and
well-refined that DNA has become, over evolutionary time, the universal information-
storage molecule for all forms of life. Nature has yet to find a better solution than
DNA for storing, expressing, and passing along instructions for making proteins.
DNA EXTRACTION
DNA extraction is a method used to purify DNA by using physical and/or chemical
methods from a sample separating DNA from cell membranes, proteins, and other
cellular components. Friedrich Miescher in 1869 did DNA isolation for the first time.
The use of DNA isolation technique should lead to efficient extraction with good
quantity and quality of DNA, which is pure and is devoid of contaminants, such as
RNA and proteins. Manual methods as well as commercially available kits are used
for DNA extraction. Various tissues including blood, body fluids, direct Fine needle
aspiration cytology (FNAC) aspirate, formalin-fixed paraffin-embedded tissues,
frozen tissue section, etc., can be used for DNA extraction.
DNA extraction involves lysing the cells and solubilizing DNA, which is followed by
chemical or enzymatic methods to remove macromolecules, lipids, RNA, or proteins.
DNA extraction techniques include organic extraction (phenol–chloroform method),
nonorganic method (salting out and proteinase K treatment), and adsorption method
(silica–gel membrane).
What does DNA extraction involve?
Step 1. Breaking cells open to release the DNA
The cells in a sample are separated from each other, often by a physical means such as
grinding or vortexing, and put into a solution containing salt. The positively charged
sodium ions in the salt help protect the negatively charged phosphate groups that run
along the backbone of the DNA.
A detergent is then added. The detergent breaks down the lipids in the cell membrane
and nuclei. DNA is released as these membranes are disrupted.
Step 2. Separating DNA from proteins and other cellular debris
To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris
as possible. This can be done by a variety of methods. Often a protease ( protein
enzyme) is added to degrade DNA-associated proteins and other cellular proteins.
Alternatively, some of the cellular debris can be removed by filtering the sample.
Step 3. Precipitating the DNA with an alcohol
Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA
sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By
gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible
and can be spooled out. If there is lots of DNA, you may see a stringy, white
precipitate.
When an ice-cold alcohol is added to a solution of DNA, the DNA precipitates out of
the solution and if there is enough DNA in the solution, you may see a stringy white
mass.
Step 4. Cleaning the DNA
The DNA sample can now be further purified (cleaned). It is then resuspended in a
slightly alkaline buffer and ready to use.
Step 5. Confirming the presence and quality of the DNA
For further lab work, it is important to know the concentration and quality of the
DNA.
Optical density readings taken by a spectrophotometer can be used to determine the
concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can
be used to show the presence of DNA in your sample and give an indication of its
quality.
APPLICATIONS OF DNA EXTRACTION
Genetic Research:
Genomic Studies: DNA extraction is crucial for studying an organism's complete set
of genes (genome) to understand genetic variations, heredity, and evolution.
Population Genetics: DNA extraction helps analyze genetic diversity within
populations, track migrations, and study the genetic basis of diseases.
Forensic Analysis:
Crime Scene Investigation: DNA extraction is used to collect and analyze DNA
evidence at crime scenes, aiding in the identification of suspects or victims.
Paternity Testing: DNA extraction is employed to determine biological relationships,
such as paternity or maternity, in legal and forensic contexts.
Medical Diagnostics:
Disease Diagnosis: DNA extraction is used to identify genetic markers associated
with various diseases, allowing for early diagnosis and personalized treatment
strategies.
Infectious Disease Testing: DNA extraction is essential for detecting and identifying
microbial DNA in clinical samples for the diagnosis of infectious diseases.
Biotechnology and Genetic Engineering:
Recombinant DNA Technology: DNA extraction is a crucial step in genetic
engineering processes, allowing the isolation of specific genes for cloning and
manipulation.
Gene Therapy: DNA extraction is involved in gene therapy research and applications
to treat genetic disorders by introducing or repairing specific genes.
Plant and Agricultural Sciences:
Crop Improvement: DNA extraction is used to study and manipulate plant genomes
for the development of genetically modified crops with improved traits, such as
resistance to pests or tolerance to environmental stress.
Biodiversity Studies: DNA extraction helps in studying the genetic diversity of plant
species, aiding in conservation efforts and sustainable agricultural practices.
Evolutionary Biology:
Phylogenetics: DNA extraction is used to analyze DNA sequences to understand
evolutionary relationships among different species and to construct phylogenetic
trees.
Personal Genomics:
Ancestry Testing: DNA extraction is utilized in direct-to-consumer genetic testing
services to provide individuals with insights into their ancestry and genetic
predispositions to certain traits or diseases.
Environmental Studies:
Ecological Research: DNA extraction from environmental samples (e.g., soil, water)
helps in studying microbial communities, biodiversity, and ecological interactions.
Archaeology and Anthropology:
Ancient DNA Analysis: DNA extraction from ancient remains allows researchers to
study the genetic makeup of extinct species and understand human evolution and
migration patterns.
Veterinary Medicine:
Animal Genetics: DNA extraction is used in veterinary medicine for studying genetic
traits, diagnosing genetic disorders, and identifying parentage in animals.
VARIOUS METHODS OF DNA EXTRACTION
Phenol–chloroform extraction
Phenol–chloroform extraction is one of the first widely used methods for
extracting DNA and is still used by many labs. Cells are first disrupted in the presence
of phenol and chloroform along with alcohol, salts, EDTA and SDS to improve the
extraction and stabilize the DNA. Subsequently the samples are centrifuged to
separate the mixture into two phases: a DNA-containing aqueous phase on the top and
an organic phase on the bottom, with a layer of cellular debris in between. After
transferring the aqueous phase to a new tube, DNA is further cleaned with ethanol
washing and precipitation.
Advantages:
Acceptable yield and purity of extracted DNA
Inexpensive
Challenges:
Very time consuming
Potential contact with harmful chemicals
High risk of cross-contamination
Requires a high level of skill avoid transferring contaminants from the solid phase
Potential for carry-over phenol, which denatures proteins
Salting out DNA
Precipitation methods with salts, such as sodium chloride, potassium acetate and
ammonium acetate, separate hydrophobic proteins and other components in the
sample from hydrophilic nucleic acid molecules. These techniques might include
cetyl trimethylammonium bromide (CTAB) to break up hard cell walls from plants,
fungi and bacteria and to help to separate polysaccharides and pigments from the
DNA. Polyvinylpyrrolidone (PVP) is a buffer component commonly added to prevent
coprecipitation of polyphenols with nucleic acids. For applications that are sensitive
to contaminants, such as PCR, extra steps for chloroform–isoamyl alcohol extraction
and ethanol precipitation is generally needed.
Advantages:
Softer than the phenol–chloroform method
Prevents hydrolysis of the bases for higher DNA integrity
Moderately easy with acceptable DNA purity
Generally low cost
Challenges:
Variable yields
Time-consuming preparation of chemicals
Potential contamination with enzyme inhibitors
Reduced yields from loss in alcohol precipitations
Solid-phase DNA extraction kits
Column-based commercial kits are quicker and easier to use than the manual
methods described above. In many of these, the negatively charged DNA binds to the
positive charge of the stationary phase of the column due to the presence of
chaotropic salts and alkaline conditions. Contaminants and high-concentration salts
are removed with centrifugation and subsequent washing steps. The DNA is eluted
from the column with nuclease-free water, often containing a buffer, such as Tris-
EDTA.
Advantages:
Very efficient and selective binding of DNA
Good quality and yield of nucleic acid
Reduced variability of yields compared to manual techniques
Reduced contamination with inhibitors in the sample
Quicker than manual methods
Challenges:
DNA length affected by shearing forces during centrifugation
Losses in yield from each column wash
Loss of DNA molecules that are too small to stick to the column
Substantial increase in plastic waste
Magnetic beads
Another method available as commercial kits is based on magnetic beads. Nucleic
acids bind to various coatings applied to small particles containing magnetic iron
oxide. Reversible binding of the nucleic acids is achieved with specific salt
concentrations. After the nucleic acids bind to the beads, the beads are held in place
with a magnet, allowing the supernatant to be removed. The nucleic acids are
subsequently released into a new buffer.
Advantages:
Fast and easy method
High-quality DNA
Option to select DNA based on size
No sheer forces from centrifugation for greater DNA integrity
Challenges:
Time-consuming tube transfers
Carry-over contamination reduces DNA quality
Requires precisely correct salt concentrations
Beads carry-over interferes with downstream applications
Single-step purification
A relatively new technique for DNA purification offers the quickest way to get
excellent DNA quality. This technique provides a unique combination of a component
that protects genomic DNA as well as active enzymes that greatly improve lysis
efficiency. In this process, the DNA flows through and everything else binds to the
column, allowing highly pure and intact genomic DNA to be obtained with only one
centrifugation step.
Advantages:
Improve performance with better purity, DNA integrity and yield
Simplify your workflow with single-step purification after lysis
Reduce time to result
Keep samples under physiological, DNA-protecting conditions
Improve lysis efficiency for great yields
Eliminate mechanical disruption and overnight lysis for non-plant samples
Avoid the risk of DNA damage from repeated centrifugation
Eliminate separate disposal of harmful chemicals
Reduce plastic waste by 70% compared to silica kits
LABORATORY PROCEDURES FOR DNA EXTRACTION
Materials and Reagents:
Biological Sample:
Depending on the source (e.g., cells, tissues, blood, bacteria), different protocols may
be required.
Buffer Solutions:
Cell lysis buffer (e.g., SDS buffer for cell disruption)
Proteinase K buffer (to digest proteins)
DNA binding buffer
Wash buffers (ethanol or isopropanol-based)
Enzymes and Proteins:
Proteinase K (digests proteins)
RNase (optional, to remove RNA)
DNA-binding proteins (to help bind DNA to the extraction column)
Salts and Other Additives:
NaCl, EDTA (for DNA precipitation and stabilization)
Isopropanol or ethanol (for DNA precipitation)
Solvents:
Phenol:chloroform:isoamyl alcohol mixture (for organic extraction, optional)
Chloroform (for organic extraction, optional)
Columns or Filters:
Silica membrane columns or magnetic beads for DNA purification.
Centrifuge Tubes and Microcentrifuge Tubes:
Sample Collection:
Collect the biological sample (cells, tissues, blood) and ensure proper storage until
extraction.
Cell Lysis:
Break down cell walls or membranes to release cellular contents.
Use a suitable lysis buffer (e.g., SDS buffer) or enzymatic digestion.
Protein Digestion:
Add proteinase K to digest proteins and RNase if needed to remove RNA
contaminants.
Incubate the mixture at an appropriate temperature for protein digestion.
DNA Precipitation:
Add salts (e.g., NaCl) and a cold alcohol (ethanol or isopropanol) to precipitate DNA.
Centrifuge to pellet DNA.
Washing:
Wash the DNA pellet with a wash buffer to remove impurities and salts.
Centrifuge and discard the supernatant.
Resuspension:
Dissolve the DNA pellet in an appropriate buffer (e.g., TE buffer).
Quantification:
Measure DNA concentration using a spectrophotometer or other quantification
methods.
Storage:
Store the extracted DNA at -20°C or -80°C for long-term storage.
CONCLUSION
DNA extraction is vital to biology, especially biotechnology. It is the first step of
different applications like fundamental research, disease diagnosis and therapeutic
decision.
One of the main advantages of DNA extraction methods is that they are very
importance to define the unique characteristics of DNA like the shape, the size and
function. DNA extraction is used in medical conditions investigation such as Down
syndrome and cystic fibrosis. It is also helpful in case of identification if a person is a
carrier of the disease or not. So, by DNA sequence in relation to diseases, It helped in
finding out the molecular basis and cure for various diseases.
In criminal investigations, DNA extraction from samples (e.g., hair – skin – blood) is
used to determine if a person is a suspect or not and also it can prove whether a person
was in the vicinity of the crime scene.
It also used in Paternity Tests. It can be useful in genetic engineering. For animals,
DNA extraction is helpful for transforming and cloning animal’s DNA. For plants,
DNA can be useful in identifying and extracting a specific gene in order to replicate
in generations of plants.DNA study also helped in creating many vaccines (e.g.,
Hepatitis B vaccine), hormones (e.g., growth hormones and insulin), and enzymes.
REFERENCES
1. Lo AC, Feldman SR. Polymerase chain reaction: Basic concepts and clinical
applications in dermatology. J Am Acad Dermatol. 1994;30:250–60. [PubMed]
[Google Scholar]
2. Clark DP, Pazdernik NJ. Molecular Biology, Polymerase Chain Reaction. 2nd ed.
United States of America (USA): Elsevier BV; 2013. pp. 163–93. Chap. 6. [Google
Scholar]
3. Niemz A, Ferguson TM, Boyle DS. Point-of-care nucleic acid testing for infectious
diseases. Trends Biotech. 2011;29:240–50. [PMC free article] [PubMed] [Google
Scholar]
4. Lorenz TC. Polymerase chain reaction: Basic protocol plus troubleshooting and
optimizing strategies. J Vis Exp. 2012;63:e3998. [PMC free article] [PubMed]
[Google Scholar]
6 QCA ( 2007) The National Curriculum Programme of Study for Science, QCA,
London.
7 M. L. Le,A. Lum-Jones,B. Saltzman,V. Visaya,A. M. Nomura,L. N. Kolonel
( 2001) Feasibility of collecting buccal cell DNA by mail in a cohort study. Cancer
Epidemiol. Biomarkers Prev. 10, 701–703.
8 L. C. Harty,M. Garcia-Closas,N. Rothman,Y. A. Reid,M. A. Tucker,P. Hartge, P.
( 2000) Collection of buccal cell DNA using treated cards. Cancer Epidemiol.
Biomarkers Prev. 9, 501–506.
9 M. Martin,M. Carrington,D. Mann ( 1992) A method for using serum or plasma as a
source of DNA for HLA typing. Hum. Immunol. 33, 108–113.
10 A,Lum,M. L. Le ( 1998) A simple mouthwash method for obtaining genomic
DNA in molecular epidemiological studies. Cancer Epidemiol. Biomarkers Prev. 7,
719–724.
