DAVANGERE UNIVERSITY

DEPARTMENT OF STUDIES IN BIOCHEMISTRY

SEMINAR TOPIC
ISOLATION, PURIFICATION, CHARACTERIZATION AND
APPLICATIONS OF NUCLEIC ACIDS
PRESENTED BY UNDER THE GUIDANCE OF
HARISH Dr. SANTOSH KUMAR M
RESEARCH SCHOLAR ASSISTANT PROFESSOR
DEPARTMENT OF BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY
DAVANGERE UNIVERSITY DAVANGERE UNIVERSITY
Contents
1. INTRODUCTION
2. Types & Sources of DNA & RNA
3. General Principles of Nucleic Acids Isolation
4. Cell Lysis Methods
5. ISOLATION OF DNA
6. Purification of DNA & RNA (Post-Isolation)
7. Characterization & Quantification of
DNA/RNA
8. Applications of DNA
9. Applications of RNA
[Link]
 [Link]
Introduction to Nucleic Acids

DNA (Deoxyribonucleic Acid): Genetic material, double helix, stores

hereditary information.
RNA (Ribonucleic Acid): Single-stranded, involved in protein synthesis
and regulation

Why Isolate DNA/RNA?

•Study genetic information and expression.
•Understand cellular mechanisms and disease pathways.
•Applications in biotechnology, medicine, forensics, agriculture.

Why Purify & Characterize?

•Ensure sample quality for downstream applications.
•Reliable sequencing, PCR, cloning, diagnostics.
 [Link] & Sources of DNA & RNA
DNA Sources RNA Sources

Total RNA (Plant, Animals, Fungi, Bacteria, Virus and

Genomic DNA (bacteria, plants, animals)
Cell cultures)
mRNA (Eukaryotic cells (plants, animals, fungi), Viruses
Plasmid DNA (Bacteria)
(RNA viruses, retroviruses)
Mitochondrial DNA (animal tissues, plant cells, and
rRNA (prokaryotic and eukaryotic ribosomes)
yeast)

Chloroplast DNA (Green leaves) tRNA (plants, animals, bacteria, yeast)

Viral DNA (Viruses) Viral RNA (Viruses)

General Principles of Nucleic Acids Isola
1. Cell Lysis: Mechanical, chemical, or enzymatic lysis
2. Removal of Proteins & Contaminants: Proteases, detergents, organic
solvents
3. Separation of Nucleic Acids: Selective precipitation or binding to
silica/resin
4. Purification: Washing to eliminate salts, polysaccharides, phenols
5. Elution: Recovery of pure DNA/RNA in buffer or nuclease-free water
6. Quality Check: Concentration & purity assessment (UV
spectrophotometer, gel electrophoresis)
 Purification Elution Quality
Source Cell Lysis Assessment
Removal of Recovery of
(Cells/Tissues/ (Mechanical, Proteins and (Washing to pure (Purity ratio,
Environmental enzymatic, or Contaminations eliminate salts, DNA/RNA in concentration,
Samples) chemical lysis) polysaccharide buffer or gel
(Proteases, s, phenols)
detergents, organic nuclease-free electrophoresi
solvents) water s)

General Principles of Nucleic Acids

Isolation
 [Link] Lysis
Methods

Mechanical Methods Enzymatic Methods

Chemical Methods
• Grinding • Lysozyme
• Detergents (SDS, Triton X-100) • cellulase
• bead beating
• chaotropic salts • proteinase K
• sonication
• solvents • lyticase
• homogenization
• freeze–thaw.
5. ISOLATION OF DNA
5.1 Genomic DNA Isolation (Bacteria)
1. Culture Preparation: Grow bacteria in LB broth overnight at 37°C.
2. Harvesting: Centrifuge 5 mL culture at 5000 rpm, 10 min.
3. Resuspension: Pellet → resuspend in TE buffer (Tris-EDTA).
• Tris maintains pH.
• EDTA chelates Mg²⁺ → inhibits DNases.
4. Cell Wall Digestion: Add lysozyme (10 mg/mL), incubate 37°C, 30 min.
5. Cell Lysis: Add SDS (1%) + Proteinase K (100 µg/mL), incubate 55°C, 1
hr.
• SDS dissolves membrane lipids.
• Proteinase K digests proteins & nucleases.
6. Extraction: Add equal volume of phenol:chloroform:isoamyl alcohol
([Link]).
• Vortex, centrifuge 12,000 rpm, 10 min.
• Aqueous phase (top layer) contains DNA.
7. Precipitation: Add 2 volumes cold ethanol + 0.1 volume sodium acetate
(3M, pH 5.2).
• Incubate –20°C, 30 min.
• Centrifuge 12,000 rpm, 15 min.
8. Washing: Wash DNA pellet with 70% ethanol.
9. Resuspension: Air-dry pellet, dissolve in TE buffer. (Wilson, 2001)
Genomic DNA Isolation (Bacteria)
DNA
5.2 Plasmid DNA Isolation (Alkaline Lysis Method)
1. Culture Preparation: Grow bacteria with plasmid overnight.
2. Harvesting: Pellet cells by centrifugation.
3. Resuspension: Resuspend in Solution 1 (Tris, EDTA,
RNase A).
• RNase removes RNA contamination.
4. Cell Lysis: Add solution 2 (NaOH + SDS).
• NaOH denatures DNA.
• SDS dissolves membranes.
5. Neutralization: Add Solution 3 (potassium acetate, pH 5.5).
• Genomic DNA & proteins form precipitate.
• Plasmid DNA remains in solution.
6. Centrifugation: Pellet contaminants, collect supernatant.
7. Purification:
• ethanol precipitation. (Birnboim & Doly, 1979)
Plasmid DNA Isolation (Alkaline Lysis Method)
5.3 Genomic DNA Isolation (Plants – CTAB Method)
1. Sample Preparation: Grind ~1 g fresh plant tissue in liquid nitrogen →
fine powder.
2. Cell Lysis: Suspend in pre-warmed CTAB extraction buffer (2% CTAB, 1.4
M NaCl, 100 mM Tris, 20 mM EDTA, 0.2% β-mercaptoethanol).
• NaCl helps dissociate DNA-protein complexes.
• β-mercaptoethanol prevents oxidation of polyphenols.
3. Incubation: 65°C for 30 min with gentle mixing.
4. Extraction: Add chloroform:isoamyl alcohol (24:1), mix gently,
centrifuge 12,000 rpm, 15 min.
• Upper aqueous phase contains DNA.
5. DNA Precipitation: Add equal volume of cold isopropanol, incubate –
20°C, 30 min.
6. Pellet Collection: Centrifuge, discard supernatant.
7. Washing: Wash pellet with 70% ethanol, air-dry.
8. Resuspension: Dissolve in TE buffer. (Doyle & Doyle, 1987)
Genomic DNA Isolation (Plants – CTAB Method)
5.4 Genomic DNA Isolation (Blood)

1. Sample Collection: Collect 1–2 mL fresh blood

(EDTA tube) or 100 mg tissue.
2. Cell Lysis: Blood: RBC lysis buffer (NH₄Cl, KHCO₃,
EDTA), centrifuge → isolate WBCs.
3. Protein Digestion: Add Proteinase K (100 µg/mL),
incubate 55°C, 1–2 hr.
4. Extraction: Add phenol:chloroform:isoamyl alcohol,
centrifuge.
5. DNA Precipitation: Add ethanol or isopropanol +
sodium acetate.
6. Washing: Wash pellet with 70% ethanol.
7. Resuspension: Dissolve DNA pellet in TE buffer.

(Miller et al., 1988)

Genomic DNA Isolation (Animal Tissues/Blood)
5.6 Mitochondrial DNA Isolation
1. Sample Preparation: Homogenize ~0.5 g tissue in isolation buffer

(250 mM sucrose, 10 mM Tris, 1 mM EDTA).

2. Centrifugation 1: 1000 g, 10 min → pellet nuclei & debris.

3. Centrifugation 2: Collect supernatant, centrifuge 10,000 g, 20 min →

pellet mitochondria.

4. Mitochondrial Lysis: Resuspend pellet in lysis buffer (Tris, EDTA,

SDS, Proteinase K).

5. DNA Extraction: Phenol-chloroform extraction.

6. DNA Precipitation: Ethanol precipitation.

(Carelli et al., 2002)

Mitochondrial DNA Isolation
5.7 Chloroplast DNA Isolation
1. Sample Preparation: Homogenize fresh green leaves in cold

isolation buffer (0.35 M sorbitol, 50mM Tris-HCl pH 7.5, 25 mM

EDTA, 0.1% BSA, 0.1 marcaptoethanol).

2. Filtration: Pass homogenate through cheesecloth → remove

debris.

3. Centrifugation: Low speed → remove cell debris.

4. Centrifugation: High speed centrifuge → collect chloroplast →

Wash.

5. Chloroplast Lysis: Resuspend chloroplasts in buffer with SDS +

Proteinase K.

6. DNA Extraction: Phenol-chloroform-isoamyl alcohol extraction.

7. Precipitation: Isopropanol precipitation → chloroplast DNA.

(Palmer, 1986)
0.35 M sorbitol
50mM Tris-HCl pH 7.5
25 mM EDTA
0.1% BSA
0.1 marcaptoethanol

100mM Tris-HCl
pH 8.0
50mM EDTA
1% SDS
100 μg/ml
Proteinase K
5.8 Viral DNA Isolation
1. Collect virus-infected cells or purified viral

particles.

2. Treat with DNase/RNase → remove host

nucleic acids.
Viral DNA

3. Lyse viral capsid using proteinase K + SDS.

4. Extract DNA with phenol–chloroform.

5. Precipitate viral DNA with ethanol.

6. Dissolve DNA in nuclease-free water or TE

buffer.
6. ISOLATION OF RNA
6.1 Total RNA Isolation
(TRIzol Method)1. Homogenize tissue/cells in TRIzol reagent (phenol +
guanidinium isothiocyanate).

2. Incubate to allow complete dissociation of

nucleoprotein complexes.

3. Add chloroform → shake vigorously → phase

separation.

4. Centrifuge → collect aqueous phase containing RNA.

5. Precipitate RNA with isopropanol.

6. Wash RNA pellet with 75% ethanol.

7. Resuspension of RNA in RNase-free water.

Total RNA Isolation (TRIzol Method)

Wash with Ethanol

6.2 mRNA Isolation (Poly-A
Selection) (Oligo-dT Magnetic
Beads)
1. Start with total RNA sample.

2. Incubate with oligo-dT beads

(complementary to Poly-A tails).

3. Wash beads → remove rRNA and

tRNA.

4. Elute bound mRNA by heating in low-

salt buffer.

5. Collect purified mRNA.

mRNA Isolation (Oligo-dT Magnetic Beads)
6.3 rRNA Isolation
Protocol (Sucrose Gradient):

1. Begin with total RNA extracted using TRIzol method.

2. Treat with DNase I → remove contaminating DNA.

3. Run total RNA through sucrose density gradient

centrifugation → separates RNAs based on size and

density.

4. Collect rRNA peaks (28S, 18S for eukaryotes; 23S,

16S for prokaryotes).

5. Purify rRNA fractions using ethanol precipitation.

6.4 tRNA Isolation
Protocol (Affinity
Chromatography):
1. Extract total RNA from tissue/cells.
2. Use size-exclusion chromatography → removes
large RNAs.
3. Apply RNA fraction to a column with specific resins
that enrich tRNA (e.g., BD-cellulose, or affinity
resins with aminoacyl-tRNA synthetases).
4. Wash away unbound RNAs.
5. Elute bound tRNA with low ionic strength buffer.
 7. Purification of DNA & RNA
(Post-Isolation)
Common Purification Methods:

•Phenol–chloroform extraction → removes proteins & lipids.

•RNase treatment → removes RNA contamination in DNA preps.

•DNase treatment → removes DNA contamination in RNA preps.

•Ethanol or isopropanol precipitation → concentrates nucleic acids.

•Column purification (silica membranes) → rapid, high-yield recovery.

8. Characterization & Quantification
of DNA/RNA
1. UV Spectrophotometry (Nanodrop/UV-Vis):
• DNA: A260/A280 ratio ~1.8 indicates purity.
• RNA: A260/A280 ratio ~2.0 indicates purity.
2. Agarose Gel Electrophoresis:
• DNA → intact, sharp bands.
• RNA → 28S & 18S bands (eukaryotic) with 28S:18S ratio ~2:1 indicates integrity.
3. Fluorometric Assays (Qubit, SYBR Green):
• Highly sensitive, specific for DNA or RNA.
4. Bioanalyzer (Capillary electrophoresis):
• RNA Integrity Number (RIN) used to assess RNA quality.
Nanodrop Spectrophotometer UV-Vis Spectrophotometer
DNA Ladders Agarose Gel Electrophoresis RNA Ladders Agarose Gel Electrophoresis
An illustration of the separation of DNA bands on a
gel (How to Read & Interpret Gel Electrophoresis, n.d.)
18s rRNA and 28s rRNA electrophoresis bands corresponding to RNA samples extracted from
culture media (Bordbar-Bonab et al., 2021)
9. Applications of DNA
 Genetic Engineering & Biotechnology –
Recombinant DNA tech, cloning, transgenic
crops.
 Forensic Science – DNA fingerprinting,
paternity testing.
 Medicine – Diagnostics (PCR tests), gene
therapy, pharmacogenomics.
 Research – Evolutionary studies, genome
sequencing, CRISPR.
 Special Sources
 Plasmid DNA → Vectors, protein
expression, DNA vaccines.
 Mitochondrial DNA → Forensics, maternal
lineage, disease studies.
 Chloroplast DNA → Plant phylogenetics,
bio-pharming.
 Etc. → Synthetic biology, personalized
medicine, nanotechnology.
10. Applications of RNA
 mRNA → Expression studies, RNA vaccines

(COVID-19).

 RNAi (siRNA, miRNA) → Gene silencing,

therapeutics.

 rRNA → Bacterial identification (16S rRNA).

 tRNA & snRNA → Protein synthesis, splicing

research.

 Diagnostics → RT-PCR for viral infections.

 Etc. → RNA editing, nanostructures, synthetic

biology.
 [Link]
• Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor Laboratory

Press.

• Green, M. R., & Sambrook, J. (2012). Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.

• Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., & Struhl, K. (2002). Short protocols in

molecular biology (5th ed.). John Wiley & Sons.

• Chomczynski, P., & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–

chloroform extraction. Analytical Biochemistry, 162(1), 156–159. [Link]

• Wilson, K., & Walker, J. (2010). Principles and techniques of biochemistry and molecular biology (7th ed.). Cambridge

University Press.

• Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2014). Molecular biology of the cell (6th ed.).

Garland Science.