Isolation, Characterization & Quality check

of Genomic DNA

Unit 1: Isolation of Genomic DNA from prokaryotic cell

13.01.25
• Differences between prokaryotic and eukaryotic cells wrt lysis of cell
and extraction of DNA
• Different methods used for isolation of genomic DNA
• Different kinds of samples used as starting points for extraction of
DNA
• Step 1. Cell lysis: To effectively release the DNA of interest into the lysate, the sample is subjected to physical
disruption methods (freezing and grinding, sonication, vortexing, bead beating) and added to a salt solution to
protect the negatively charged phosphate groups in the DNA backbone. A non-ionic detergent such as sodium
dodecyl sulfate (SDS) may also be added to break down the lipids in the cell membrane and nucleus
• Step 2. Removal of cellular debris: To free the sample from unwanted materials such as DNA-associated proteins,
cellular proteins, and other cellular debris. This is accomplished through filtration, centrifugation, bead-based
methods, or by adding the appropriate protease to the solution
• Step 3. Precipitating the DNA: After the removal of unnecessary debris, the DNA is precipitated by carefully
adding ice-cold ethanol or isopropyl alcohol and centrifuging the sample solution. Presence of alcohol and salt
renders the DNA insoluble, adding monovalent cations such as ammonium or sodium acetate to the DNA solution
encourages efficient nucleic acid precipitation while allowing the proteins to remain in the organic phase
• Step 4. Purification: The recovered DNA pellet is washed with cold alcohol and centrifuged to remove all
contaminants (e.g., proteins, salts). It is then re-suspended in a slightly alkaline elution buffer such as Tris, TE, or
double-distilled water
• At this stage, the DNA sample is ready to use
• Step 5. Confirming the presence of DNA: The presence and quality of the DNA sample can be confirmed through
DNA electrophoresis on an agarose gel and checking it under UV light
• Cells with soft cell walls (e.g., bacteria) can be lysed by simply heating the sample solution, while cells with
harder cell walls (e.g., plant cells, algae, fungus) require a combination of physical (or mechanical), chemical, and
enzymatic methods. On the other hand, animal cells are best lysed using the phenol-chloroform method
 Cell wall composition
Plant: Cellulose
Bacteria: Peptidoglycan
Fungi: Chitin
Animal: Absent

• Genomic DNA: The comprehensive set of

genetic instructions housed within the
nucleus of eukaryotic cells and the main
body of prokaryotic cells.
• Plasmid DNA: Smaller and circular, existing
independently within bacterial cells
• Fragment DNA: Smaller pieces of DNA
often used in genetic analysis and research
• Mitochondrial DNA: Carries genes for
energy production located within the
mitochondria
• Chloroplast DNA: Carries genes for
photosynthesis located in the chloroplasts
• Prokaryotic DNA and Microorganisms DNA
• Prokaryotic DNA is essential for a wide range of applications in microbial
research and industrial biotechnology, including genetic engineering,
sequencing, and microbial diversity studies
• Prokaryotic DNA is typically more straightforward to extract than eukaryotic
DNA due to the absence of a nuclear membrane and the simpler cell
structure
• Cell lysis: The presence of robust cell walls in many prokaryotes, such as
Gram-positive bacteria require lysis techniques to ensure efficient DNA
release
• Bacteria, yeast, and fungi have tough cell walls that require mechanical
disruption (e.g., bead beating) or enzymatic treatment (e.g., lysozyme for
bacteria, zymolyase for yeast) for effective lysis before extraction
• Eukaryotic DNA
• mtDNA is typically smaller and circular, which may require gentle handling to
avoid shearing. Use mild conditions during cell lysis to avoid damage.
 Cell lysis

Physical/
Chemical Enzymatic
Mechanical

*Most of the times combination methods are used

 Physical Methods
• Typically involve some type of sample grinding or crushing to disrupt the cell walls or tough
tissue
• A common method of physical disruption is freezing and grinding samples with a mortar and
pestle under liquid nitrogen to provide a powdered material that is then exposed to chemical or
enzymatic lysis conditions
• Physical methods are often used with more structured input materials, such as tissues or plants
• Other devices use bead beating or shaking in the presence of metallic or ceramic beads to
disrupt cells or tissues, or sonication to disrupt tissues and lyse cells.
 Chemical Methods
• Can be used alone with easy-to-lyse materials, such as tissue culture cells or in combination with
other methods
• Chemicals commonly used include detergents (e.g., SDS) and chaotropes (e.g., guanidine salts
and alkaline solutions).
 Enzymatic Methods
• Often used with more structured starting materials in combination with other methods with
tissues, plant materials, bacteria and yeast
• Help to disrupt tissues and tough cell walls
• Depending on the starting material, typical enzymatic treatments can include: lysozyme,
 S.NO. Physical means Chemical Means Enzymatic Means Toxins
What • Use of rotating blades to • Often used in conjunction • Provide a non- Only for eukaryotic
grind and disperse large with mechanical grinding mechanical cells
amounts of complex • Break the lipid barrier method for cell
tissue surrounding cells by lysis

How • Cell membrane is • By solubilizing proteins • Cleave the cross • Selectively

physically broken down • Disrupting lipid-lipid, links and branching hemolytic and has
by shear or external protein-protein, and networks a preference for
forces protein-lipid interactions rabbit red blood
cells
• Upon binding self-
oligomerization
occurs, resulting in
hexameric
aggregates
believed to be
transmembrane
pores
Benefits • Efficient • Rapid • Efficient
• Control over buffers used • Gentle • High lysing
• High lysing efficiency • Efficient, and efficiency
reproducible

Disadvant • Localized heating within a • Buffer components may • Buffer components

ages sample can need to be removed may need to be
• Requires equipment before downstream removed
 Sonicator Bead beating

Grinder

Mortar and Pestle

 Methods for genomic DNA isolation

• Three general methods for genomic DNA isolation are common, and each is
based on a different biochemical principle
• Organic extraction and precipitation
• Genomic DNA isolation by organic extraction involves the addition of phenol and guanidine
isothiocyanate to separate the DNA and proteins into different organic phase
• Organic extraction is a low-cost , is a straightforward method requiring very little equipment
• Silica
• DNA binds to silica (glass fibers) under high-salt conditions and can be released under low-salt conditions
• Silica-containing columns provide an easy way to bind, wash, and elute purified genomic DNA
• Columns are designed to flow buffers through centrifugation, vacuum, or gravity
• Paramagnetic beads
• Paramagnetic (attracted to magnet) beads are added to the sample, and genomic DNA binds to the beads
• Using a strong magnet, the beads are held in place while removing unwanted material
• After washing, the genomic DNA is eluted from the beads in water or a low-salt buffer
• The bead-based method is scalable and automation compatible
 DNA extraction methods
• The DNA extraction methods are broadly categorized into chemical and mechanical methods,
• Chemical DNA extraction methods:
• Organic DNA extraction
• Phenol-chloroform method
• Inorganic DNA extraction
• Proteinase K DNA extraction
• CTAB DNA extraction
• SDS DNA extraction
• Salting out method
• Silica-gel-based techniques
• Physical DNA extraction methods
• Magnetic bead DNA extraction
• Paper DNA extraction
 Chemical DNA extraction

• Also known as liquid or solution-based DNA extraction

• Highly relies on different chemicals
• Thus, extensive chemical preparation is required
• The organic solvent-based separation uses organic chemicals, for example, phenol
and chloroform
• The inorganic solvent-based techniques include other chemicals like SDS, CTAB,
NaCl and MgCl2
 Phenol-chloroform method

• Type: Chemical/liquid separation technique

• Subtype: Solution-based (liquid-liquid) organic DNA
extraction
• Purity: Excellent
• Yield: Excellent
• Sample: Animal, plant, fungi, bacteria or even
plasmid
• Tissue type: Blood, saliva, solid tissue, plant leaf or
root, cell culture, skin or hair
Phenol digest proteins, isoamyl alcohol
separate nucleic acid and chloroform reduce
the foaming between interphase
Advantages Disadvantages
• Unsafe and health hazardous
• Requires extensive and tedious chemical
• Excellent quality and yield preparation
• The technique is cheap, easy and reliable
• Time-consuming and requires training and/or
experience to prepare and handle chemicals
 CTAB DNA extraction (Cetyl Trimethyl Ammonium Bromide)

• Other chemicals like SDS,

• Type: Chemical/liquid separation technique Beta-mercaptoethanol, PVP
• Subtype: Solution-based organic DNA extraction and Triton X 100 are also
combined
• Category: Liquid-liquid separation • CTAB and SDS remove glycoproteins
• Purity: Excellent and the rest of the chemicals remove
• Yield: Excellent polyphenolic compounds
• Cellulase and pectinase, etc are often
• Sample: Plant sample combined with the technique to
• Tissue type: Plant leaf, bark, dead plant parts and root remove harf polysaccharides of the
plant cell wall

Advantages Disadvantages
• Requires extensive chemical preparation
• The CTAB buffer removes • Time consuming and tedious
polysaccharides and polyphenols
effectively • Requires additional techniques like
tissue homogenization and the use of
• Gives excellent yield for plant DNA liquid nitrogen
 Proteinase K DNA extraction

• Type: Chemical DNA extraction • Enzymatic catalytic reaction to

• Subtype: Enzymatic technique neutralize, unfold and digest
nuclear and cellular proteins
• Purity: Excellent
• Yield: Excellent
• Sample: Any biological sample

Advantages Disadvantages
• Easy and Safe
• Provides high purity and yield
• Accurate and rapid • A bit costly
• Can be used for any sample • Additional SOP to store and process
proteinase K is needed
• Can be combined with any protocol
• Requires a small sample volume
 Spin-column DNA extraction
• Chemical method of DNA extraction but works on the
• Type: Chemical DNA extraction principle of solid-phase separation
• Subtype: Solid-liquid phase DNA • A silica gel as a solid phase is immobilized in a tube
extraction method • Lysis buffer is used to lyse the sample and subsequently allow it to
• Purity: Excellent separate on a solid phase
• Centrifugation first removes all the debris by protecting the DNA and
• Yield: Good elutes DNA in the last step by changing the pH of the solution
• Sample: Any biological sample • Such spin-columns and lysis buffer are nowadays commercially available

Advantages Disadvantages
• Fast, accurate, easy to perform and safe
Isolates high-quality DNA • Costly
•
Doesn’t require precipitation • Lacks optimizations
•
Works for small volume samples • The yield remains comparatively low but good
•
 DNA extraction by chromatography

• Not used recently

• Type: Physical separation technique
• Subtype: Solvent-based DNA extraction
• Purity: Decent
• Yield: Decent

Advantages Disadvantages
• Low yield and quality
• Easy to perform • Can’t differentiate between DNA and
• Comparatively rapid RNA (IOC)
 DNA extraction by CsCl density gradient centrifugation

• Type: Physical separation technique

• Subtype: density gradient centrifugation
• Purity: Not good
• Yield: Not good

Advantages Disadvantages
• Can isolate non-supercoiled, circular, plasmid • Tedious, time-consuming & unsafe
and bacterial DNA. • Low yield and quality.
 Magnetic bead-based DNA extraction

• Magnetically charged beads made up of sulfate and hydroxyl

• Type: Physical DNA extraction
groups, and DNA binding antibodies are used to separate DNA
• Subtype: Solid phase DNA extraction • Beads are mixed with DNA and allowed to separate in a high
• Purity: Excellent magnetic field
• The bead-bound DNA will remain on the bottom while debris
• Yield: Excellent comes out in the supernatant. The beads are washed and DNA
• Sample: Any biological sample eluted. In the final step, the DNA detached from the beads by
heating at 60 to 65°C temperature

Advantages Disadvantages
• Fast (15-20 min), accurate, safe & reliable
• Doesn’t require centrifugation and extensive • Additional magnetic instrument setup is
chemical preparation required
• Automated
 Paper DNA extraction

• Type: Physical DNA extraction • When a drop of sample is applied to the

cellulose paper, treated with a washing buffer
• Subtype: – Solid-liquid DNA and resuspended into the elution buffer, DNA
extraction can be extracted
• Purity: -N/A
• Yield: -N/A
• Sample: – N/A

Advantages Disadvantages
• Not as effective as other assays
• Fast, easy to use, cost-effective and safe • Requires large samples
 Different kinds of samples used as starting points for extraction of DNA
• Genomic (gDNA)
• Cell-free (cfDNA)
• FFPE
• Prokaryotic DNA
• Tissues for forensic studies (Buccal swab, urine, hair, and
blood samples)
• Plasmid
• Mitochondrial (mtDNA)
• Chloroplast (cpDNA)
• Blood
• Viruses