This presentation gives an overall idea about the DNA, its history, protocol for isolation, methods of quantification and different storage conditions

1. DNA- Basics on Isolation,
Quantification, Storage
B. Naveen Rajeshwar
AAH-PB0-06

2. Nucleic acids
 Biopolymers, or large biomolecules, essential to all known forms of life
 Composed of nucleotides- The monomers
 Nucleosides- 5-carbon sugar + nitrogenous base
 Nucleotides- 5-carbon sugar + phosphate group + nitrogenous base
 Two main classes of nucleic acids are
 Deoxyribonucleic acid (DNA)- Deoxyribose sugar
 Ribonucleic acid (RNA)- Ribose sugar

3.  Nucleobases- Purine, pyrimidine
Purine- adenine & guanine (2 rings)
pyrimidines - cytosine & thymine (DNA)
(1 ring) cytosine & Uracil in (RNA)
 Sugar + phosphate- backbone
“phosphodiester bond”
 Sugar + nucleobase- “N-glycosidic
linkage”

4. DNA- Why so important?
• DNA encodes all the information needed by every cell to function and thrive.
• DNA carries hereditary information (copied and passed intact over generations)
• A gene is a segment of DNA.
• Genetic code- Biochemical instructions found within most genes
• Genetic code specify the chemical structure of a particular protein.
• DNA structure of a gene determines the arrangement of amino acids, the type & function
of the protein manufactured.

5. History- The chemistry of genes
First DNA extraction attempt was performed by Friedrich Miescher in 1869.
Isolated the cell material from pus cells (WBC) in waste surgical bandages (Accidental isolation)
Named it the “nuclein” later on his student named it, a “nucleic acid”.
By 1930s, P. Levene, W. Jacobs, and others had demonstrated that
RNA = sugar (ribose) + 4 nitrogenous bases
DNA = different sugar (deoxyribose) + 4 bases & each base is coupled with sugar-phosphate to form nucleotide.
In 1958, Meselson and Stahl developed a full-function protocol for DNA extraction.
Density gradient centrifugation method was the 1st protocol used for isolating E.coli DNA.
Proteinase K enzyme method of DNA extraction- Lahiri and Nurnberger in 1991 (reported earlier
by Miller et al., in 1988)
Phenol-chloroform isoamyl alcohol method- Joseph Sambrook and David W. Russell (most popular-
yield, purity & consistency are pretty decent)

6. DNA Extraction/ Isolation
 Simplest explanation- “extracting DNA from cells”.
 DNA extraction or DNA 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.
 Other common definitions
“Isolating DNA by disrupting cell wall/cell membrane and a nuclear membrane”
“Isolating nucleic acid from rest of the cell organelle”
“Isolation of DNA by breaking the cell membrane and nuclear membrane with the help of
chemicals, enzyme or physical disruptions”

7. DNA isolation- Purpose
o Main purpose- To provide pure, unfragmented & highly concentrated DNA for DNA testing
o Direct bodily cells or tissue can not be used
o DNA testing- DNA profiling, heritage testing, diagnosis, PCR, RFLP, blotting, hybridisation
o Isolation of DNA is needed for genetic analysis- Scientific, medical, or forensic purposes.
o Scientists use DNA in a number of applications, such as
Introduction of DNA into cells and animals or plants
Diagnostic purposes (most common)
Identification of individuals (Crime victims)
Paternity determination
Organism identification.
o Why pure DNA required? Impurities interfere with DNA analysis methods & reduce quality &
storage life.

8. Sources
 Sources for DNA isolation are very diverse.
 Basically it can be isolated from any living or dead organism.
 Common sources- whole blood , hair, sperm , bones, nails, tissues, blood stains, saliva ,
buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria,
animal tissues, or plants.
 Stored samples- Archived tissue samples, frozen blood or tissue, exhumed bones or tissues,
and ancient human, animal, or plant samples.
 Methods used to isolate DNA are dependent on the source, age and size of the sample.
Cell type Organism Methods
Soft cell wall Some bacteria Heating, simple lysis buffer
Hard cell wall Plants, some fungi, algae Mechanical- chemical- enzymatic methods
Cell membrane Animal cell Enzymatic and chemical method

9. Basic procedure
• Isolation of DNA basically consists of four major steps.
i. Preparation of a cell extract.
ii. Purification of DNA from cell extract.
iii. Concentration or precipitation of DNA samples.
iv. Quantification or Measurement of purity
• The isolated DNA is stored for future use

11. • To extract DNA from a tissue/cells of interest, the 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. So, the
same types of chemicals can work for both.
Cell Lysis
Mechanical disruption
 Grinding- mortar &
pestle
 Adding lysis buffer
Enzymatic disruption
 proteinase K
 Peptidase
 Protease
Disrupt proteins by
digesting them.
Chemical disruption
 SDS, CTAB, salts
 Tris, detergents
Lyse cell wall/ cell
membrane by
solubilizing them
After cell lysis, debris are removed by centrifugation
1. Preparation of a cell extract

12. • In addition to DNA the cell extract will contain signifiant 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.
• The most commonly used procedures are:
i. Phenol–chloroform extraction.
ii. Inorganic enzymatic extraction
iii. Minicolumn purification
2. Purification of DNA from cell extract

13. Phenol–chloroform extraction
• Phenol–chloroform extraction in which phenol denatures proteins in the sample.
• After centrifugation of the sample, denaturated proteins stay in the organic phase while aqueous
phase containing nucleic acid is mixed with the chloroform.
• Later precipitated by ethanol and dissolved in TE buffer
Enzymatic method of DNA extraction
• The enzyme like proteinase K, CTAB are used to denature the protein following the cell lysis by
ectraction buffer.
• Sample is boiled with enzymes at 55-60°C followed by centrifugation and precipitation by ethanol
and sodium acetate.
• DNA is then dissolved in TE buffer
Minicolumn purification
• Minicolumn purification that 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.

14. Ethanol precipitation
• The most frequently used method of concentration is ethanol precipitation.
• Ethanol precipitation usually by ice-cold ethanol or isopropanol [OH + water= H2 bond]
• Precipitation of DNA is improved by increasing of ionic strength, usually by adding sodium
acetate. [Sodium ions acts with phosphate ions of DNA= complex]
• DNA is insoluble in alcohol, it will aggregate together, giving a pellet upon centrifugation.
• In a concentrated DNA solution, a glass rod can be used to pull out the adhering DNA strands.
• It is called as spooling of DNA
• Dilute solutions precipitated DNA can be collected by centrifugation and redissolving in an
appropriate volume of nuclease free water/ TE buffer.
3. Concentration or precipitation of DNA samples

16. • Quantification of nucleic acids is done to determine the average concentrations 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.
• Various methods that can be employed to quantify the nucleic acid concentration are listed below,
UV Spectrophotometry
Nanodrop
Ethidium Bromide Staining
Gel Electrophoresis Analysis
Real time PCR
Bioanalyzer
Fluorometric Quantification
 PicoGreen
 Qubit
 Hoechst 33258 dye
4. Quantification of nucleic acids

17. UV Spectrophotometry
 Biomolecules absorb light in UV range.
 Allows us to estimate amount of DNA by its absorbance
 DNA: 260nm and 280nm [Purines & pyrimidines- absorbance maxima at 260 nm]
 Proteins: between 215-230nm [peptide bonds] & 280nm [Aromatic AA]
 Both proteins and DNA absorb light at 280nm. If sample is mixed, this can interfere with one
another
 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

18.  A ratio of <1.8 signifies contamination with protein or phenol & the preparation is not
proper
 DNA purity: A260/A280 ratio: 1.8
 DNA concentration (μg/ml): 50 μg/mL (OD value 1at 260 nm)
 DNA yield: DNA conc * Total volume of DNA solution
The relationship between concentration of DNA, RNA, Protein and absorptivity:
Sample Absorbance value Quantity (approximate)
Double-stranded DNA 1at 260 nm 50 μg/mL
Pure single-stranded DNA 1 at 260 nm 33 μg/mL
Pure RNA 1 at 260 nm 40 μg/mL
Pure protein (vary in general) 1 at 280 nm 1 mg/mL

19. • Absorbance of solution at two
wavelengths ( 260nm and 280nm)
• Calculate ratio A260/A280
• Ratio of less than 1.8 signifies that sample
is contaminated with protein or phenol.
Indications poor extraction.
• Dependent on pH and ionic strength of
buffer
• Absorbance of solution at two
wavelengths ( 260nm and 230nm)
• Calculate ratio A260/A230
• If ratio varies, may indicate presence of
residual phenol, magnetic beads,
carbohydrates.
Nucleic Acid Purity determined by:
Pure RNA: 2.0
Pure DNA: 1.8
Prue Protein: 0.6
Pure RNA/DNA: 2.2-1.8
UV Spectrophotometry: Nanodrop

20. Advantages:
 Uses small microvolumes (1-2 μl)
 Rapid results for quick assessments
 Graph gives indication of quality
 Widely used
 concentration range from 2-15000 ng/μl can
be assessed
 Automatic pathlength adjustment
 quantification of concentrated samples
avoiding the need for sample dilutions
Disadvantages:
 Bad resolution for low concentration samples
(lower limit of 2ng/ul)
 Does not distinguish between ds or ssDNA
 Contaminating samples leads to falsely high
quantitation readings

21. Negative values: dirty
pedestals or incorrect blank
Ragged line: Bad blank
Jagged line: Broken read or low
volume
High 230nm: Contaminates;
carbohydrates, phenols, Guanidine
isothiocyanate

22.  Binds to Nucleic Acid and gives orange fluorescence under UV radiation from 500 – 590
nm.
 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
Advantages:
• Specific bands
• Not pure samples
Disadvantages:
• Need lots of DNA
• Not very accurate
Ethidium Bromide Staining

23. o Uses fluorescent dye
o PicoGreen- binds dsDNA
o Measure fluorescent intensity of PicoGreen dye with spec.
o DNA quantified by comparing sample to set of standards
o Qubit- binds DNA, RNA or protein depending on kit
• Similar Advantages and Disadvantages to PicoGreen
o Hoechst 33258 dye- specific to DNA
• Good for both large and small amounts of DNA
Advantages:
 High throughput
 Increased sensitivity
 Less prone to contaminants
Disadvantages:
 Need special equipment and reagents/kit
 Longer prep time
Fluorometric Quantification

24. • 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 0.01–15 μg/mL 0.1–10 μg/ml 0.025– 1000 ng/mL
RNA Not applicable 1–40 μg/mL Minimal sensitivity

25. Real time PCR
Fluorescent dye binding to dsDNA as it accumulates during PCR process
Targets specific region of DNA template
Used for sequencing prep, to verify quality and quantity of DNA libraries
Bioanalyzer- automated electrophoresis
֍ Size, quantitation and purity assessments
֍ Small volume of sample needed
֍ Multiple platforms (RNA, DNA)
֍ Both low and high concentration samples
֍ Easy to use, but expensive
Hybridization-Based Techniques
ᴥ Southern or Northern Blotting
ᴥ “Probe sequence” based
ᴥ Higher resolution (down to actual nucleotide sequence)
Other quantification methods

26. Storage of DNA
Main threat- Contamination with nucleases & chemical degradation
 DNA storage methodologies depends on- type of DNA, duration of storage, storage temperatures &
conditions and downstream applications
 There are four temperature based strategies: -20° C, -80° C, -196° C, Dried, at room temperature
Major drawback- repeated freeze & thaw cycles have a deleterious effect on the quality of the DNA.
Storage at vitreous state
 Glassy state- lying between the solid and liquid states
 At -196° C, DNA is maintained in a vitreous state [No nuclease activity & 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 drying

27. Medium length storage
• Storage of DNA for medium term is done at -20° C or -80° C
• 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 -80° C)
• Yet, ethanol must be removed prior to use (Undesirable technique)
Short term storage
ᴥ Often used samples of DNA can be stored in aliquots at 4° C to avoid repeated freeze-thaw
cycles.
ᴥ Kept in TE buffer- stable for 6 to 12 months
ᴥ Regularly monitored for DNA concentration and evaporation

28. References
• https://www.news-medical.net/life-sciences/How-to-Store-DNA.aspx
• Nishiguchi, M. K., Doukakis, P., Egan, M., Kizirian, D., Phillips, A., Prendini, L., ... & Giribet, G. (2002). DNA isolation procedures. In Techniques in
molecular systematics and evolution (pp. 249-287). Birkhäuser, Basel.
• William, S., Feil, H., & Copeland, A. (2012). Bacterial genomic DNA isolation using CTAB. Sigma, 50(6876).
• https://www.mlsu.ac.in/econtents/281_DNA%20quantification.pdf
• https://nptel.ac.in/content/storage2/courses/102103013/pdf/mod4.pdf
• https://nptel.ac.in/content/storage2/courses/102103013/pdf/mod3.pdf
• https://www.pacb.com/wp-content/uploads/2015/09/SharedProtocol-Extracting-DNA-usinig-Phenol-Chloroform.pdf
• Gaikwad, A. B. (2020). DNA storage methodologies: Principles and
Protocols.http://www.nbpgr.ernet.in/Portals/6/DMX/GENOMIC_RESOURCES/DNA%20storage%20methods-principles%20and%20protocols.pdf
• Dhaliwal, A. (2013). DNA extraction and purification. Mater Methods, 3, 191.
• https://www.slideshare.net/AYESHAKABEER3/methods-of-dna-extraction
• Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (No. Ed. 2). Cold spring harbor laboratory press.
• https://www.colorado.edu/ecenter/sites/default/files/attachedfiles/seracare_stability_of_genomic_dna_at_various_storage_conditions_isber2009.pdf
• Pirttilä, A. M., Hirsikorpi, M., Kämäräinen, T., Jaakola, L., & Hohtola, A. (2001). DNA isolation methods for medicinal and aromatic plants. Plant
Molecular Biology Reporter, 19(3), 273-273.
Thank you..!