DNA Isolation Reviewer

DNA Isolation Reviewer (MLS 420)

1. KEY DEFINITIONS

DNA Extraction — collecting DNA by physical or chemical means.
DNA Purification — eliminating contaminants (proteins, lipids) from extracted DNA.
DNA Isolation — the complete process: extraction (cell lysis) + purification.

2. MOLECULAR BIOLOGY WORKFLOW

Sample Collection → Nucleic Acid Extraction → Quantification & Purity Check (NanoDrop) / Quality Check (Gel Electrophoresis) → Amplification (PCR) → Gel Electrophoresis → Downstream Applications

3. BASIC NUCLEIC ACID ISOLATION STEPS

Step

Purpose

Cell Lysis

Break cell to release NA from nucleus

Separation

Remove proteins and other cell material

Purification

Wash away unwanted material

Concentrate

Increase NA concentration (optional but important for PCR/sequencing)

4. STEP 1: CELL LYSIS / DISRUPTION METHODS

Mechanical

  • Mortar and pestle

  • Vortexing with beads

  • Sonication — uses ultrasonic waves

  • High-pressure homogenizer / French press — sudden pressure change ruptures cells

Chemical

  • CTAB (Cetyltrimethylammonium bromide) & SDS (Sodium Dodecyl Sulfate) — dissolve membrane

  • β-mercaptoethanol — breaks disulfide bonds in proteins

  • EDTA — inhibits nucleases

  • Tris buffer (pH 8) — maintains pH

  • NaCl — stabilizes nucleic acid

Enzymatic

  • Proteinase K → animal cells

  • Cellulase → plant cells

  • Lyticase → yeast

  • Lysozyme → bacteria

Thermal — heat-based disruption

5 Factors Influencing Disruption Strategy: Stability of molecules, size of sample, cohesion of cells, cell membrane type, presence of inhibitors.

5. STEP 2.1: DENATURATION & SEPARATION FROM BIOMOLECULES

  • Chemical: SDS, Phenol-Chloroform, protein denaturation

  • Enzymatic: Protease/Proteinase K

  • Centrifugation — separates by density

    • Aqueous phase (top) = RNA/DNA

    • Interphase = DNA

    • Organic phase (bottom) = proteins/lipids

6. STEP 2.2: PRECIPITATION OF NUCLEIC ACIDS

  • Monovalent cations: Ammonium, Potassium, Sodium Acetate, Lithium, or NaCl

  • Precipitating agents: 95% Ethanol (absolute) or Isopropanol

  • Followed by centrifugation

7. STEP 3.1: WASHING THE PELLET

  • Use 70–80% Ethanol

  • Centrifuge to collect purified pellet

  • Removes remaining salts and impurities

8. STEP 3.2: DRYING & DISSOLVING THE PELLET

  • Air dry or vacuum dry (ethanol is volatile)

  • Dissolve in molecular grade water or TE buffer

  • Optional: heat at 50–55°C water bath to facilitate dissolution

9. NUCLEIC ACID STABILIZATION

  • Resuspend in TE buffer (neutral pH, chelating agent)

  • Store at 5°C or -70°C

  • Aliquot samples

  • AVOID freeze-thaw cycles

  • Use nuclease-free, MolBio-grade plasticware

10. POST-ISOLATION STEPS

  • Isolated DNA → apply RNase treatment

  • Isolated RNA → apply DNase treatment

  • Gel Electrophoresis — checks integrity and fragment size

  • NanoDrop — checks quantity and purity

11. NUCLEIC ACID ISOLATION METHODS Chemical-Based

Method

Key Points

Phenol-Chloroform (GTPC)

Organic solvent separates NA from proteins/lipids; results in aqueous, interphase, organic layers

Alkaline Extraction

NaOH + SDS lyse bacteria; used for plasmid DNA; chromosomal DNA and proteins precipitate after neutralization

CTAB Extraction

Used for plant cells; CTAB binds polysaccharides to remove them

EtBr-CsCl Gradient

Uses ultracentrifugation + ethidium bromide; separates DNA by density; visualized under UV light

Solid-Phase Methods

Uses spin columns, magnetic beads, or automated systems. 4 steps: Lysis → Binding → Washing → Elution

3 Principles:

  1. Size exclusion (gel filtration) — large molecules elute first; small molecules enter pores and elute later

  2. Ion-exchange chromatography — DNA (negatively charged) binds to positively charged matrix; eluted by changing salt concentration or pH

  3. Affinity chromatography — DNA binds silica under high salt; impurities washed away; eluted with low salt buffer or water

12. SOURCES OF NUCLEIC ACID

Microorganism

Human Sample

Virus, Bacteria, Fungi, Parasites

Blood, Saliva, Tissues, Hair, Skin, Stool, Other body fluids

13. AFTER DNA ISOLATION

  • Have identifiable, quantifiable genomic DNA

  • Ready for downstream applications (PCR, sequencing, cloning)

  • Both quality AND quantity matter

QUICK RECALL — COMMON EXAM HITS

  • CTAB & SDS → dissolve membranes

  • EDTA → inhibits nucleases

  • β-mercaptoethanol → breaks protein disulfide bonds

  • Lysozyme → bacteria; Proteinase K → animal cells

  • Centrifugation principle → density

  • Washing step → 70–80% ethanol

  • Storage → TE buffer, 5°C to -70°C

  • Avoid → freeze-thaw cycles

  • CTAB extraction → plant DNA

  • Alkaline extraction → plasmid DNA from bacteria

  • NanoDrop → quantity and purity

  • Gel electrophoresis → integrity and size