Genomic DNA Isolation.pptx

Genomic DNA Isolation
&
DNA Quantification
Dr Sarath Krishnan M P
Junior Resident/Biochemistry
AIIMS Rishikesh
22/08/2022 1
Dr Sarath Krishnan M P/JR-2/Bchem

Learning Objectives!!!!
• Introduction and History
• What are the purpose of DNA extraction???
• What all samples???
• What are the different methods???
• What are the four steps of DNA extraction???
• How we store the extracted DNA???
• Newer developments
22/08/2022 Dr Sarath Krishnan M P/JR-2/Bchem 2

Molecular Biology
• Genetics!!!!
Application of genetics to medical practice
A. Studies of the inheritance of disease in families.
B. Mapping of disease genes to specific locations on chromosomes
C. Analysis of the molecular mechanisms through which genes cause disease
D. Diagnosis and treatment of genetic disease (ex. Gene therapy)

Nucleic
Acid
DNA
Genetic
Material
RNA

DNA-Why So Important
• Encodes all the information needed by
every cell to function
• Carries hereditary information
• GENE
• Genetic code
• Arrangement of amino acids, the type
and function of the protein manufactured

DNA Extraction/ Isolation
• Simplest explanation: “extracting DNA from cells”
• DNA extraction/Isolation is a process to isolate or obtain high-quality
DNA from biological samples
• DNA isolation: Process of purification of DNA from sample using a
combination of physical and chemical methods

Why pure DNA required????

History
• First DNA extraction attempt : Friedrich Miescher in 1869
• Waste surgical bandages- Pus cells(WBC)- Accidental isolation
• “Nuclein”- “Nucleic acid”
• By 1930s, P.Levene, W.Jacobs, demonstrated
• RNA=Sugar(Ribose)+4 nitrogenous base
• DNA=Deoxyribose+4 bases and each base is coupled with sugar-phosphate
to form nucleotide.

History(Contd....)
• In 1958, Meselson and Stahl developed a full-function protocol for DNA
extraction
• Density gradient centrifugation method : 1st protocol for isolating E.coli DNA
• Proteinase K enzyme method of DNA extraction-Lahiri and Nurnberger in 1991
• Phenol-Chloroform isoamyl alcohol method- Joseph Sambrook and David W.
Russell(most popular-yield, purity and consistency are pretty decent)

DNA isolation- Purpose
• Main purpose: To provide pure, unfragmented and highly
concentrated DNA.
• DNA testing: DNA profiling, Genetic diagnosis, PCR, RFLP,
Blotting, Hybridisation
• Isolation of DNA is needed for genetic analysis: Scientific, Medical
or Forensic purposes

DNA isolation- Purpose(Contd....)
DNA use in a number of applications, such as
A. Introduction of DNA into cells (animals or plants)
B. Diagnostic purposes(most common)
C. Identification of individuals(Crime victims)
D. Paternity determination
E. Organism identification

What all Samples for DNA Extraction????
• Isolated from any living/ dead organism
Common sources
Whole blood, Hair, Sperm, Bones, Nails, Tissues, Blood stains
Saliva, Buccal swabs, Epithelial cells, Urine
Paper cards used for sample collection
Bacteria
Animal tissues or Plants

What all Samples for DNA Extraction????
Stored samples
Archived tissue samples, Frozen blood or tissue
Exhumed bones or tissues
Ancient human, animal or plant samples
• Methods used to isolate DNA are dependent on the source, age and size of the
sample

Methods of DNA Extraction
LIQUID PHASE
• Organic(Phenol-Chloroform)
extraction
• Non-organic(Proteinase K and
Salting out) extraction
SOLID PHASE
• Silica based method
• Magnetic bead method
• Chelex (Ion exchange resin)
extraction

Soft Cell wall
• Bacteria
• Heating, Simple
lysis buffer
Hard Cell
wall
• Plants, Fungi,
Algae
• Mechanical-
Chemical-
Enzymatic
methods
Cell
membrane
• Animal cell
• Enzymatic and
chemical
method
How do we decide???

Steps of DNA extraction
Isolation of DNA basically consists of four major
steps
A. Cell Lysis: Disruption of cell membrane
B. Removal of cellular proteins
C. Precipitating the DNA with ethanol
D. Quantification and purity measurement of DNA
• The isolated DNA is stored for future use

Step A: Cell lysis-Disruption of the cell
membrane
• To extract DNA : Cellular components have to be separated and the
cell membranes have to be disrupted
• The nuclear membrane and cell membrane are made up of protein
and lipids : Same types of chemicals can work
• After cell lysis, debris are removed by centrifugation

Cell lysis
Mechanical
disruption
*Grinding-mortar
and pestle
*Adding lysis
buffer
Enzymatic
disruption
*Proteinase K
*Peptidase
*Protease
Chemical
disruption
*SDS, CTAB,
Salts
*Tris

Composition of Buffers
RBC Lysis Buffer
• NH4Cl 8.29g [155mM]
• KHCO3 1g [10mM]
• Na2EDTA 0.034g or 200μl
EDTA 0.5M [0.1mM]
• Fill to 1000ml with distilled
water
• Adjust to pH 7.4 with 1M HCl or
NaOH for each use.
Proteinase K (10mg/ml)
• Dissolve 100mg Proteinase K in
10ml TE for 30min at room
temperature
• Aliquot and store at -20֯C
Detergent
• SDS
• Triton-X 100

Functions of the Buffer components
• EDTA - Chelates Mg2+ ions (Essential for the action of nucleases)
• Tris buffer - Low ionic strength environment (Better DNA solubility in water)
• Proteinase K - Degradation of cellular proteins for easy removal. Nucleases
are broken down and rendered non effective
• SDS/ Triton-X - Detergents denature proteins which can be then better acted
upon by Proteinase K

Step B: Removal of cellular proteins
• In addition to DNA the cell extract will contain significant quantities
of detergents, proteins, salts and reagents used during cell lysis step
• A variety of procedures can be used to remove these contaminants,
leaving the DNA in a pure form

Step B: Removal of cellular
proteins(Contd....)
• The most commonly used procedures are:
1. Phenol-chloroform extraction
2. Non-organic (Proteinase K and Salting out) extraction
3. Minicolumn purification

Phenol-chloroform extraction
• Phenol-chloroform extraction in which pheol denatures proteins in
the sample
• Phenol:Chloroform:Isoamyl alcohol=25:24:1
• After centrifugation of sample, denatured proteins stay in the
organic phase while aqueous phase containing nucleic acid.

Phenol-chloroform extraction(Contd....)
• Later precipitated by ethanol and dissolved in TE buffer

Role of the various components(Phenol -
chloroform extraction)
• Phenol - Removes proteins.
• Isoamyl alcohol—Acts as an antifoaming agent.
• Chloroform - Solubilizes lipids and removes excess phenol.
• 70% Ethanol - Removes excess salts.
• Isopropanol / Ethanol - Precipitates DNA.
• TE Buffer

Phenol-chloroform extraction(Contd....)
ADVANTAGES
• Most effective at extracting large
amounts of DNA
• Used on a wide range of samples
DISADVANTAGES
• Slow
• Labour-intensive
• Toxic(Phenol, Chloroform)

Non-organic (Proteinase K and Salting
out) extraction
• Cell membranes are lysed and proteins are denatured using Proteinase K,
CTAB or SDS
• RNA is removed with RNase
• Sample is incubated with enzymes at 55-60֯C
• Proteins are precipitated with salt solution(Ammonium acetate, Sodium
Chloride)
• DNA is precipitated by ethanol and dissolved in TE buffer

Non-organic (Proteinase K and Salting
out) extraction [Contd....]
ADVANTAGES
• Fast and easy method
• Uses nontoxic materials, no
fume hood required, no
hazardous materials disposal
issues
• Produces high-quality DNA
DISADVANTAGES
• If salts are not adequately
removed, problems could occur
with the RFLP procedure due to
alteration of DNA mobility (band
shifting

Minicolumn purification
• Main principle behind Kit based
methods
• Relies on the fact that the
nucleic acids may bind
(adsorption) to the solid
phase(silica or other) depending
on the pH and the salt
concentration of the buffer

Minicolumn purification(Contd....)
ADVANTAGES
• Quick
• Highly purified DNA
DISADVANTAGES
• Multiple sample transfer gives an
increased risk of contamination

Magnetic bead method
• Magnetic beads are coated with DNA antibodies or silica to bind with DNA.
A. Samples are lysed & and then treated with proteinase K.
B. The lysates are then applied to the beads and external magnetic field is
applied
C. Wash
D. Magnetic beads are separated from the sample on a magnetic stand using
paramagnetic effect
E. DNA is eluted

Magnetic bead method(Contd....)
ADVANTAGES
• Very fast, may be automated
• Highly purified DNA
• Excellent for liquid blood
DISADVANTAGES
• Cannot be used directly on stain
i.e. need to remove cells from stain
substrate (cloth, etc.)
• Very expensive

Other methods of DNA extraction
A. Chelex (Ion exchange resin) extraction
B. FTA paper method
Fig A Fig B

Step C:Precipitating the DNA with ethanol
• Most frequently : Ethanol precipitation
• Ethanol precipitation : Ice-cold ethanol/ isopropanol
• Precipitation of DNA is improved by increasing of ionic strength,
usually by adding sodium acetate
• DNA is insoluble in alcohol, it will aggregate together, giving a pellet
upon centrifugation

Step C:Precipitating the DNA with ethanol
(Contd....)
• In a concentrated DNA solution, a glass rod can be used to pull out the
adhering DNA strands
• Spooling of DNA
• Dilute solutions precipitated DNA can be collected by centrifugation
and redisolving in an appropriate volume of nuclease free water/ TE
buffer

Protocol of DNA extraction: Non-organic
(Proteinase K and Salting out) extraction
1. Dispense 450 μl of RBC Lysis Solution into a 1.5 ml microcentrifuge tube
2. Add 150 μl, whole blood and mix.
3. Incubate for 5 min, at room temperature (15–25°C).
4. Centrifuge for 2 mins at 13,000–16,000 x g, to pellet the WBC
5. Carefully discard the supernatant, leaving ~10 μl, of the residual liquid and the
WBC pellet.
6. Add 150 μl, Cell Lysis Solution, vortex for 10sec

[Contd....]
7. Add 50 μl, Protein Precipitation Solution, vortex for 20 s
8. Centrifuge for 2 min at 13,000–16,000 x g, the precipitated proteins form a dark
brown pellet.
9. Add 300 μl isopropanol into 1.5 ml tube, and add the supernatant from the
previous step .
10. Mix by inverting gently 50 times until the DNA is visible as threads or a clump.
11. Centrifuge for 1 min at 13,000–16,000 x g
12. Discard the supernatant (Take care the pellet)

[Contd....]
13. Add 300 μl, of 70% ethanol and invert several times to wash the DNA pellet.
14. Centrifuge for 1 min at 13,000–16,000 x g, carefully discard the supernatant
(Air dry the pellet for 5 sec)
15. Add 50 μl, DNA Hydration Solution and vortex for 5 s
16. Incubate at 65°C for 5 min, to dissolve the DNA.
17. Incubate at room temperature overnight with gentle shaking.

Importance of salt in DNA precipitation
with ethanol
• The protease solution already contains salt.
• Na+ ions of NaCl bind to the phosphate groups of
DNA molecules and neutralizing the charge
• The addition of NaCl allows the DNA molecules to
come together.
• Reduces the hydrophilic nature of DNA

Precautions during DNA extraction
A. Gentle handling to avoid shearing of the DNA
B. Sterile tubes and tips
C. Sterile buffer and solution
D. Avoid using glass test tubes and glass rods.
E. Avoid DNA contamination from other sources

Points to be noted....
• High-molecular-weight DNA is vulnerable to hydrodynamic shearing
forces because of minimal lateral stability.
• Hydrodynamic flow
• The longer the DNA molecule, the weaker the force required for
breakage.

Step D: Quantification and purity
measurement of DNA
• Determine the average
concentration of DNA or RNA
present in a mixture, as well as
their purity
• The accurate measurement is
based on sensitivity, specificity
and interference by contaminants

Methods of Quantification
UV Spectrophotometry - Nanodrop
Ethidium Bromide Staining - Gel electrophoresis analysis
Fluorometric quantification - Pico Green/ Qubit/ Hoechst 33258 dye
Bioanalyzer

Beer- Lambert’s Law

UV spectrophotometry
• Biomolecules absorb light in UV range
• Allows us to estimate amount of DNA by its absorbance
• DNA: 260nm and 280nm[Purines and pyrimidines- Absorbance maxima at
260nm]
• Proteins: Between 215-230nm[Peptide bonds] and 280nm[Aromatic AA]
• Both proteins and DNA absorb light at 280nm, If sample is mixed, this can
interfere with one another

UV spectrophotometry(Contd....)
• Calculating the ratio of A260/A280 gives purity of sample
• Value of this ratio is 2.0, 1.8 and 0.6 for pure RNA, DNA and protein respectively
• A ratio of <1.8 signifies contamination with protein or phenol and the preparation is not
proper
• DNA purity: A260/A280 ratio=1.8
• DNA concentration(μg/ml): 50μg/ml (OD value 1 at 260nm)
• DNA yield: DNA Conc x Total volume of DNA solution

Relationship between concentration of
DNA, RNA, Protein and absorptivity
Sample Absorbance value Quantity
(Approximate)
dsDNA 1 at 260nm 50μg/ml
Pure ssDNA 1 at 260nm 33μg/ml
Pure RNA 1 at 260nm 40μg/ml
Pure protein(Vary in
general)
1 at 280nm 1mg/ml

UV Spectrophotometry: Nanodrop
• Absorbance of solution at two wavelengths (230, 260 and 280nm)
• A260/A280
• Dependent on pH and ionic strength of buffer
• Ratio of <1.8 signifies that sample is contaminated with protein or phenol - Indicates
poor extraction
• A260/A230 - >2 (>3 is not acceptable)
• If ratio varies, may indicate presence of residual phenol, magnetic beads, carbohydrates.

UV spectrophotometry:
Nanodrop(Contd....)

Advantages
• Uses small microvolumes
• Rapid result
• Graph gives indication of quality
• Concentration range from 2-
15000ng/μl can be assesed
• Automatic pathlength adjustment
Disadvantages
• Bad resolution for low
concentration samples (lower
limit of 2ng/μl)
• Does not distinguish between ds
or ssDNA
• Contaminating samples leads to
falsely high quantitation readings

How to solve this????
• A DNA extraction resulted in 50μl total volume. The sample was
diluted 1:5 and gave an OD260 reading of 0.307 units.
i. dsDNA Conc???
ii. DNA yield???

Answer!!!!
i. dsDNA Conc= 50μg/ml x OD260 reading x Dilution factor
= 50 x 0.307 x 5
= 76.75μg/ml
ii. DNA yield= dsDNA conc/ 1000ml x Total sample volume
= 76.75/1000 x 50μl
= 3.83μg

Ethidium bromide staining
• Binds to nucleic acid and gives orange fluorescence under UV radiation from
500-590nm
• EtBr - A potent mutagen and carcinogen
• Gel electrophoresis analysis
• Calculate band size using software from imager[Gel Doc]
• Compare fluorescence intensities of ladder and sample to estimate DNA
concentration

Ethidium bromide staining(Contd....)

Ethidium bromide staining(Contd....)
Advantages
• Specific bands
Disadvantages
• Need lots of DNA
• Not very accurate

Fluorometric Quantification
• Use fluorescent dye
• PicoGreen - Binds dsDNA
• Measure fluorescent intensity of PicoGreen dye which binds with
DNA
• DNA quantified by comparing sample to set of standards

Fluorometric Quantification(Contd....)
Advantages of PicoGreen
• High throughput
• Increased sensitivity
• Less prone to contaminants
Disadvantages of PicoGreen
• Need special equipment and
reagents/ Kit
• Longer preparation time

• Hoechst 33258 dye - Specific to DNA
- Good for both large and small amounts of DNA
• Quantification of nucleic acids separated by gel electrophoresis
• Done by comparing the stained nucleic acids with stained standards of known
concentration separated on the same gel
• The dye: DNA complex shows greater fluorescence than the unbound dye

• The fluorescence intensity of the band estimates the concentration/
amount of DNA
Nucleic Acid Fluorescence methods
Hoechst 33258 EtBr PicoGreen
DNA 0.01-15μg/ml 0.1-10μg/ml 0.025-
1000ng/ml
RNA NA 1-40μg/ml Minimal
sensitivity

QuBit fluorometer
• Binds to DNA, RNA or protein
• Kit Based
• Advantages and disadvantages
same as of Pico Green

Other Quantification methods
Bioanalyzer
• Quantification and purity assessments
• Small volume of sample needed
• Multiple platforms (DNA, RNA)
• Both low and high concentration samples
• Easy to use, but expensive

Storage of DNA
• Main threat - Contamination with nucleases and chemical degradation
• DNA storage methodologies depends on - type of DNA, duration of storage,
storage temperatures and conditions and downstream applications
• There are four temperature based strategies: -20֯C, -80֯C, -196֯C, Dried, at
room temperature
• Major drawback - Repeated freeze and thaw cycles have a deleterious effect
on the quality of the DNA

Storage of DNA(Contd....)
Long term storage
• Glassy state - Lying between the solid and liquid states
• At -196֯C, DNA is maintained in a vitreous state[No nuclease activity and
chemical degradation - long term storage - over decades]
• Dry state - Dehydrated DNA stored at room temperature
Should be kept at low humidity [Moisture can cause hydrolytic reactions]
Done by spray drying, spray freeze dying

Medium length storage
• Storage of DNA for medium term is done at -20֯C or -80֯C
• Can prevent degradation for months or years
• Acidic conditions cause hydrolysis of DNA
• DNA in the aqueous phase is stored under slightly basic conditions (Tris:EDTA
buffers used - pH 8.0)
• Can be stored as precipitate under ethanol (at -80֯C)
• Yet, ethanol must be removed prior to use(Undesirable technique)

Short term storage
• Often used samples of DNA can be stored in aliquots at 4֯C to avoid
repeated freeze-thaw cycles
• Kept in TE buffer - Stable for 6 to 12 months
• Regularly monitored for DNA concentration and evaporation

Newer developments in DNA extraction

*Paul R, Ostermann E, Wei Q. Advances in point-of-care nucleic acid extraction technologies for rapid diagnosis of
human and plant diseases. Biosensors and Bioelectronics. 2020 Dec 1;169:112592.
Fig A Fig B Fig C
Fig D Fig E Fig F

Fig A
Fig A: Pandoh PK, Corbett RD, McDonald H, Alcaide M, Kirk H, Trinh E, Haile S, MacLeod T, Smailus
D, Bilobram S, Mungall AJ. A high-throughput protocol for isolating cell-free circulating tumor DNA
from peripheral blood. Biotechniques. 2019 Feb;66(2):85-92.
**Yan YY, Guo QR, Wang FH, Adhikari R, Zhu ZY, Zhang HY, Zhou WM,
Yu H, Li JQ, Zhang JY. Cell-free DNA: hope and potential application in
cancer. Frontiers in Cell and Developmental Biology. 2021 Feb 22;9:639233.

MGISP NE-32 robot Mgi
https://biodiagnostico.com.uy/en/hospital-de-florida-incorpora-robot-de-mgi-para-extraccion-
automatizada-de-adn-arn/

Overview of Genomic DNA extraction

References
• Nishiguchi MK, Doukakis P, Egan M, Kizirian D, Phillips A, Prendini L, Rosenbaum HC, Torres
E, Wyner Y, DeSalle R, Giribet G. DNA isolation procedures. InTechniques in molecular
systematics and evolution 2002 (pp. 249-287). Birkhäuser, Basel.
• William S, Feil H, Copeland A. Bacterial genomic DNA isolation using CTAB. Sigma.
2012;50(6876).
• Gaikwad AB. DNA storage methodologies: Principles and Protocols.
• Dhaliwal A. DNA extraction and purification. Mater Methods. 2013;3:191.
• Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold spring harbor
laboratory press; 1989.
• Pirttilä AM, Hirsikorpi M, Kämäräinen T, Jaakola L, Hohtola A. DNA isolation methods for
medicinal and aromatic plants. Plant Molecular Biology Reporter. 2001 Sep;19(3):273
• Biochemistry manual AIIMS Rishikesh (UG&PG)

Thank you & Have a NICE day

Genomic DNA Isolation.pptx

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