DNA Isolation: A Complete CSIR-NET Guide (Concepts, Steps & Exam Traps)

DNA isolation (also called DNA extraction) is one of the most fundamental techniques in molecular biology and a frequently tested topic in CSIR-NET, GATE, DBT-JRF, and university exams. Questions are asked not just on steps, but on the role of each reagent, principle, and experimental variations.

This blog explains DNA isolation step-by-step, with exam-oriented explanations and high-yield facts.

What is DNA Isolation?

DNA isolation is the process of separating DNA from cellular components such as:

• Cell wall
• Cell membrane
• Proteins
• RNA
• Lipids
• Polysaccharides

The final goal is to obtain pure, intact DNA suitable for downstream applications like:

• PCR
• Restriction digestion
• Cloning
• Sequencing
• Southern blotting

Principle of DNA Isolation (VERY IMPORTANT FOR CSIR-NET)

The principle is based on:

1. Cell lysis
2. Removal of proteins and contaminants
3. Precipitation of DNA

DNA is:

• Negatively charged
• Insoluble in alcohol (ethanol/isopropanol)
• Stable in slightly alkaline pH

General Steps of DNA Isolation

Cell Lysis (Breaking the Cell)

The first step is to break the cell wall and membrane to release DNA.

Sample Type	Method Used
Bacteria	Lysozyme + detergent
Plant cells	Grinding + CTAB
Animal cells	Detergents only

 Detergents used:

• SDS (Sodium dodecyl sulfate)
• CTAB (Cetyl trimethyl ammonium bromide)

CSIR-NET Tip:
CTAB is especially used for plant DNA isolation because it removes polysaccharides.

Removal of Proteins

After lysis, DNA is mixed with proteins (histones, enzymes).

 Common methods:

• Phenol–chloroform extraction
• Proteinase K digestion

Phenol–Chloroform Method

• Phenol → denatures proteins
• Chloroform → improves phase separation
• Centrifugation forms two layers:

Layer	Contains
Upper aqueous phase	DNA
Lower organic phase	Proteins

 Exam Trap:
DNA remains in aqueous phase, not organic phase.

 Removal of RNA

RNA contamination is removed using:

• RNase A

RNase is:

• Heat stable
• Does not require cofactors

DNA Precipitation

DNA is precipitated using alcohol.

Alcohol Used	Condition
Ethanol (cold)	2–2.5 volumes
Isopropanol	0.6–1 volume

 Salt required:

• Sodium acetate / NaCl

Why salt?
Neutralizes negative charge on DNA phosphate backbone.

CSIR-NET Favorite Question:
DNA precipitates because it is insoluble in alcohol.

Washing and Resuspension

• Wash DNA pellet with 70% ethanol
• Air dry
• Dissolve in:
• TE buffer (Tris-EDTA)
• Nuclease-free water

Role of EDTA:

• Chelates Mg²⁺
• Inhibits DNases

CTAB Method for Plant DNA (High-Yield Topic)

Plant cells contain:

• Polysaccharides
• Polyphenols

CTAB:

• Forms complexes with polysaccharides
• Prevents DNA degradation

β-mercaptoethanol is added to:

• Remove polyphenols
• Prevent oxidation

CSIR-NET Repeated Concept:
CTAB is preferred for plant genomic DNA isolation.

Quality and Quantity Check of DNA

UV Spectrophotometry

Ratio	Interpretation
A260/280 ≈ 1.8	Pure DNA
< 1.8	Protein contamination
> 2.0	RNA contamination

 DNA absorbs at 260 nm, proteins at 280 nm.

 Agarose Gel Electrophoresis

• Intact DNA → sharp high-molecular-weight band
• Smearing → degraded DNA

 Common CSIR-NET Exam Traps

✔ Phenol is corrosive
✔ EDTA inhibits DNase
✔ Isopropanol requires less volume than ethanol
✔ CTAB removes polysaccharides
✔ DNA is negatively charged
✔ RNase removes RNA, not DNA

One-Line CSIR-NET Memory Box

·         Lysozyme → bacterial cell wall

·         SDS → membrane lysis

·         Phenol → protein denaturation

·         RNase → RNA removal

·         Cold ethanol → DNA precipitation

·         EDTA → DNase inhibition

NA isolation is the process of extracting pure DNA from cells by removing membranes, proteins, and RNA.

Main Steps:

1.      Cell lysis – Detergents (SDS/CTAB) break cell membrane and wall.

2.      Protein removal – Phenol–chloroform or Proteinase K removes proteins.

3.      RNA removal – RNase digests RNA.

4.      DNA precipitation – Cold ethanol/isopropanol + salt precipitates DNA.

5.      Washing & resuspension – 70% ethanol wash; dissolve in TE buffer.

Key Exam Points:

·         CTAB → used in plant DNA isolation (removes polysaccharides).

·         EDTA → inhibits DNase by chelating Mg²⁺.

·         DNA precipitates in alcohol because it is insoluble.

·         Pure DNA A260/280 ≈ 1.8.

 CSIR-NET tip: DNA stays in the aqueous phase during phenol–chloroform extraction.

Conclusion

DNA isolation is not just a laboratory technique but a conceptual goldmine for CSIR-NET. Understanding:

• Why each reagent is used
• Which step removes what
• How purity is measured

will help you confidently solve assertion-reason, match-the-following, and numerical questions in the exam.

 

DNA Isolation – CSIR NET MCQ Quiz

1. The main purpose of EDTA in DNA isolation buffer is to:

Precipitate DNA
Denature proteins
Chelate divalent cations
Lyse cell membrane

2. Which detergent is commonly used to lyse cell membranes during DNA isolation?

SDS
Agarose
EDTA
Tris-HCl

3. Proteinase K is used in DNA isolation to:

Degrade RNA
Degrade proteins
Precipitate DNA
Chelate Mg²⁺ ions

4. Which reagent is used to remove RNA contamination?

RNase A
DNase I
SDS
Chloroform

5. DNA is precipitated commonly using:

Phenol
Ethanol or Isopropanol
Tris buffer
SDS

6. Phenol–chloroform extraction mainly removes:

DNA
RNA
Proteins and lipids
Salts

7. High salt concentration during DNA precipitation helps in:

DNA degradation
Protein solubilization
Neutralizing DNA charges
RNA digestion

8. A 260/280 ratio of ~1.8 indicates:

RNA contamination
Protein contamination
Pure DNA
Degraded DNA

9. Which enzyme degrades DNA if not inhibited during isolation?

RNase
Ligase
DNase
Polymerase

10. Tris-HCl buffer maintains:

Osmotic balance
pH stability
DNA precipitation
Protein digestion

The Molecular Word Processor: The Next Step Beyond CRISPR

CRISPR is a powerful gene-editing tool that works like molecular scissors. It can cut DNA and help scientists edit genes. But CRISPR has one problem: it makes a full cut in the DNA, and sometimes the cell repairs this cut in the wrong way. This can lead to mistakes.

Because of this, scientists have created newer and safer gene-editing tools. These new tools work like a “Molecular Word Processor” instead of scissors. They can edit DNA without making big cuts. This makes gene editing more accurate and less risky.

CRISPR-Cas9: The Molecular Scissors

CRISPR-Cas9 was the first major gene-editing tool.

How it works:

• A guide RNA finds the place in the DNA that needs editing.
• The Cas9 enzyme cuts both strands of the DNA.
• The cell repairs the cut, but sometimes the repair is messy or wrong.

This method is powerful but not always safe because it creates a double-strand break.

Base Editing: The Molecular Pencil

Base Editing was the next improvement. It does not cut the whole DNA. Instead, it changes just one letter of the DNA.

How it works:

• A modified Cas9 makes a small nick in one DNA strand.
• An enzyme changes one DNA base to another (like C to T or A to G).

Why it matters:
Many diseases are caused by a single wrong DNA letter. Base editing can fix these “DNA typos” safely.

Prime Editing: The Molecular Word Processor

Prime Editing is the most advanced tool so far. It works like the “Find and Replace” feature in a computer.

It can:

• Change any DNA letter
• Insert small DNA sequences
• Delete small DNA sequences
   all without making a dangerous double-strand cut.

Prime Editing uses three parts:

1. Nickase Cas9 – cuts only one DNA strand
2. Reverse Transcriptase – writes the new DNA
3. pegRNA – guides the editor and provides the corrected DNA sequence

Steps:

1. The pegRNA guides the editor to the target.
2. Nickase Cas9 cuts one strand.
3. The Reverse Transcriptase writes the new DNA.
4. The cell replaces the old DNA with the new, corrected version.

This method is clean, safe, and very accurate.

Why Prime Editing Is Important

Prime Editing can do:

• Substitutions (change one base)
• Insertions (add small pieces)
• Deletions (remove small pieces)

It can fix around 89% of known genetic mutations.
And it does this without making a full cut in the DNA.

Future Tools: Transposons and TIGR

Scientists are working on even newer systems:

1. Transposon-based editors
   These may help insert large pieces of DNA, like whole genes.
2. TIGR systems
   These are very small proteins that may edit any part of the genome easily.

These tools could make gene editing even more powerful and flexible.

Prime Editing and Sickle Cell Disease

Sickle Cell Disease happens because of one small mistake in the HBB gene.

• Healthy DNA: GAG
• Sickle DNA: GTG

This single-letter change causes red blood cells to become sickle-shaped.

Prime Editing can correct GTG back to GAG by changing just one base.

Why it is better than CRISPR-Cas9:

• CRISPR makes a full cut; Prime Editing makes only a small nick
• Prime Editing is more accurate
• Prime Editing causes fewer mistakes

How treatment works:

1. Doctors collect the patient’s blood stem cells.
2. They edit the cells using Prime Editing.
3. The corrected cells are placed back into the patient.
4. These cells can make healthy red blood cells for life.

This could be a permanent cure.

The Future of Gene Editing

CRISPR started the revolution.
Base Editing made it cleaner.
Prime Editing made it precise.

Soon, gene editing may cure diseases, help in drug discovery, and prevent genetic problems before they occur.

We are entering a future where DNA can be edited as easily as editing words in a document.

Table: Major Fields of Biotechnology and Their Key Applications

Biotech Field	Key Focus	Important Applications	Examples
Medical Biotechnology	Developing therapies & diagnostics	Vaccine development, gene therapy, disease diagnostics	mRNA vaccines, CRISPR-based therapies
Agricultural Biotechnology	Crop improvement & protection	High-yield crops, pest-resistant plants, stress-tolerant varieties	Bt Cotton, Golden Rice
Industrial Biotechnology	Large-scale bio-processing	Enzyme production, biofuels, bioplastics	Bioethanol, biodegradable plastics
Environmental Biotechnology	Pollution control using microbes	Wastewater treatment, bioremediation, bio-filtering	Oil spill clean-up, heavy metal removal
Food Biotechnology	Food quality & preservation	Fermentation, probiotic foods, nutritional enhancement	Yogurt, cheese, fortified foods
Marine Biotechnology	Exploring marine organisms	Novel enzymes, pharmaceuticals, bioactive compounds	Anticancer compounds from algae
Microbial Biotechnology	Using microbes for innovation	Antibiotics, enzyme production, fermentation industries	Penicillin, lactase enzyme

 

Advanced Biotechnology Quiz

15 MCQs for CSIR-NET / GATE / DBT-JRF practice. Choose one answer for each question, then click Submit.

Q1. In Sanger sequencing, which molecule acts as a chain terminator causing termination of DNA synthesis?

A. dATP B. ddNTP (dideoxynucleotide) C. rNTP D. DNA ligase

Q2. Which of the following is FALSE about quantitative PCR (qPCR) using SYBR Green dye?

A. SYBR Green binds to double-stranded DNA. B. SYBR Green can differentiate between specific and non-specific products. C. Melt curve analysis helps identify primer-dimers. D. SYBR Green fluorescence increases proportionally with product amount.

Q3. In enzyme kinetics, which plot is used to determine Vmax and Km by linearization?

A. Michaelis-Menten plot (v vs [S]) B. Lineweaver-Burk plot (1/v vs 1/[S]) C. Scatchard plot D. Hill plot

Q4. Which method is most suitable for isolating high-molecular-weight genomic DNA free from RNA contamination?

A. Boiling lysis without RNase B. CTAB extraction followed by RNase treatment C. Phenol-only extraction D. Direct PCR from colony

Q5. In plant tissue culture, which plant growth regulator combination is commonly used to induce callus formation?

A. High cytokinin, low auxin B. High auxin, low cytokinin C. No hormones D. Gibberellin alone

Q6. Which technique is used to measure genome-wide DNA methylation at single-base resolution?

A. ChIP-seq B. ATAC-seq C. Bisulfite sequencing D. RNA-seq

Q7. In next-generation sequencing, 'paired-end' reads are useful because they:

A. Reduce sequencing error rate to zero B. Provide sequence information from both ends of a DNA fragment, helping with alignment across repeats C. Double the read length automatically D. Only used in Sanger sequencing

Q8. Which statement about Agrobacterium tumefaciens-mediated transformation is CORRECT?

A. It is effective only in monocots and not dicots B. It transfers T-DNA from the Ti plasmid into the plant genome C. It uses electroporation to enter plant cells D. It inserts RNA directly into the nucleus

Q9. Which of the following best describes a 'silent mutation' at the codon level?

A. Changes an amino acid to a stop codon B. Changes the DNA sequence but not the encoded amino acid C. Always causes a frameshift D. Deletes an exon

Q10. In fermentation, which parameter is most directly used to estimate microbial biomass in real time?

A. Optical density (OD) at 600 nm B. pH measurement C. Glucose concentration D. CO2 sensor only

Q11. Which DNA repair pathway is primarily active during non-dividing (G0/G1) cells and is error-prone?

A. Homologous recombination (HR) B. Base excision repair (BER) C. Non-homologous end joining (NHEJ) D. Mismatch repair (MMR)

Q12. Which viral vector is most commonly used for stable integration into dividing mammalian cells for gene therapy?

A. Adeno-associated virus (AAV) B. Adenovirus C. Retrovirus / Lentivirus D. Bacteriophage

Q13. Which technique separates proteins based on isoelectric point and molecular weight sequentially?

A. SDS-PAGE B. 2D gel electrophoresis C. Western blot D. Size-exclusion chromatography

Q14. In population genetics, which effect describes loss of genetic variation when a new population is founded by a small number of individuals?

A. Bottleneck effect B. Founder effect C. Gene flow D. Stabilizing selection

Q15. Which reagent is commonly used as a reducing agent to break disulfide bonds during SDS-PAGE sample preparation?

A. SDS B. β-mercaptoethanol (or DTT) C. EDTA D. Triton X-100

Tip: This quiz is for practice. For exam-style preparation, attempt without notes and time yourself for 20 minutes.