Nucleic Acid Isolation Reviewer

Nucleic Acid Isolation Reviewer

WHAT IS NUCLEIC ACID ISOLATION?

It is the most crucial step in molecular biology. It involves two processes combined:

Extraction — breaking/lysing the cell to release nucleic acid from the nucleus Purification — removing contaminants (proteins, lipids, polysaccharides) so only DNA or RNA remains

GOAL: Pure, good quality (intact, not degraded), and sufficient quantity of nucleic acid

Three types of isolation:

• DNA isolation only

• RNA isolation only

• Both DNA and RNA

WORKFLOW IN MOLECULAR BIOLOGY

Sample Collection → Nucleic Acid Extraction → Quality/Quantity Checking → Amplification (PCR) → Gel Electrophoresis → Downstream Applications

SOURCES OF NUCLEIC ACID

PRETREATMENT AND WEIGHING

Some samples need pretreatment before cell lysis:

• Tissues in paraffin → Deparaffinization and rehydration

• Bones and teeth → Decalcification (remove minerals, soften sample)

• Plants → Grinding (strong cell wall)

Weighing — heavier sample = more nucleic acid expected

CHOOSING A METHOD

Consider these three factors:

1. Efficiency and cost — Manual methods are cheap but tedious and hazardous; commercial kits are fast but expensive

2. Sufficient quantity for downstream applications (e.g., PCR has a required DNA amount)

3. Purity of the final nucleic acid extract

USES OF NUCLEIC ACID EXTRACTION

• Scientific Research — DNA/RNA studies in disease, biotechnology

• Medical — identifying etiology of infections, drug resistance, disease progression prediction

• Forensic — paternity testing, suspect identification

GENERAL STEPS FOR NUCLEIC ACID ISOLATION

STEP 1: CELL LYSIS (Tissue Homogenization / Cell Disruption)

Goal: Break the cell membrane → penetrate to nucleus → release nucleic acid

A. Mechanical Methods

Use mechanical methods only for plant/fungal cells. Human cells have thin membranes — chemicals/enzymes are enough.

B. Chemical Methods

C. Enzymatic Methods

D. Thermal Method Uses heat alone to break open cells. Simple and common — used in Colony PCR.

Factors that Influence Cell Disruption Strategy:

• Stability of molecules (RNA isolation → cold conditions to inactivate RNAse)

• Size of sample (large → more aggressive mechanical disruption)

• Cohesion of cells (tightly bound tissues like skin/muscle are harder to break)

• Cell membrane type (thick walls need stronger methods)

• Presence of inhibitors (e.g., heme in RBCs inhibits PCR → use plasma instead)

STEP 2: SEPARATION (Denaturation of other biomolecules from NA)

Two techniques: Centrifugation and Precipitation

Chemical Treatment:

• Phenol — denatures proteins, separates NA from proteins

• Chloroform — increases density of organic layer (makes contaminants sink to bottom)

• SDS — assists protein denaturation

Enzymatic Treatment:

• Protease/Proteinase K

Centrifugation produces 3 layers:

• Aqueous phase (top) — contains RNA and DNA

• Interphase — may contain DNA

• Organic phase (bottom) — contains proteins, lipids (contaminants)

→ Aqueous phase is collected for precipitation.

Precipitation (Monovalent Cations + Alcohol):

• Salts used: Ammonium, Potassium, Sodium Acetate, Lithium Chloride, NaCl

• Alcohol used: 95% Ethanol (absolute) or Isopropanol

• Salt neutralizes negative charge of phosphate backbone → promotes DNA aggregation

• Nucleic acids are not alcohol-soluble → they precipitate and form a pellet

STEP 3: PURIFICATION (Washing + Drying)

Washing:

• Use 70–80% ethanol (has 20–30% water to dissolve and wash away excess salts)

• Wash pellet at least twice

• Centrifuge after each wash

• Do NOT over-wash (nucleic acid may be lost)

Drying:

• Air drying or vacuum drying

• Removes excess alcohol (alcohol evaporates)

• Do NOT over-dry (DNA will break without moisture)

• Dry for 2–3 minutes then resuspend immediately

Resuspension:

• For DNA → use TE buffer (Tris-EDTA) to maintain pH and stability

• For RNA → use nuclease-free water to prevent degradation

STEP 4: CONCENTRATE (Optional)

• Done when sample is light (low expected yield)

• Before cell lysis → concentrates both DNA and RNA

• After cell lysis → concentrates either DNA or RNA specifically

NUCLEIC ACID ISOLATION METHODS

A. CHEMICAL-BASED METHODS (Traditional)

1. Organic Isolation — INVOLVES phenol and chloroform

• Very efficient but hazardous

• Subtypes: Phenol-chloroform extraction, GTPC extraction

GTPC (Guanidinium-Thiocyanate-Phenol-Chloroform) Extraction:

• Most common for RNA isolation

• Cell lysis uses Guanidinium isothiocyanate

• Separation uses phenol and chloroform

• Two options: Isoamyl alcohol or TRIzol reagent

Organic Method Pros and Cons:

2. Inorganic Isolation (Salting Out Method) — does NOT use phenol and chloroform

• Uses Sodium Acetate instead

• Sodium → reduces negative charge of DNA, stabilizes DNA

• Acetate → acts as buffer, neutralizes pH change

3. Alkaline Method

• Uses alkaline conditions (NaOH) to disrupt cells and denature proteins

• Commonly used for plasmid DNA extraction from bacteria

4. CTAB Extraction

• Very strong detergent for plant and fungal cells

5. Cesium Chloride Gradient Centrifugation (with Ethidium Bromide)

• Inorganic method; hazardous (EtBr is carcinogenic)

• EtBr used as dye to visualize colorless nucleic acids

• CsCl is the alternative to phenol/chloroform

B. SOLID-PHASE METHODS ("Next Generation Isolation Method")

Uses solid particles (columns, beads) to facilitate isolation.

4 Basic Steps:

1. Lysis — break the cell

2. Binding — nucleic acids bind to immobilized reagent/medium

3. Washing — remove contaminants

4. Eluting — separate nucleic acids from the immobilized medium

Principles:

1. Size Exclusion by Gel Filtration

• Uses beads with pores

• Micromolecules get trapped in pores → delayed elution

• Macromolecules pass through edges → eluted first

2. Ion Exchange Chromatography (Anion Exchange)

• Immobilized medium has positive charge

• Attracts negatively charged nucleic acids

• Contaminants are washed away

3. Affinity Chromatography

• Uses specific ligands that only bind DNA or RNA

• Based on molecular specificity

NUCLEIC ACID STABILIZATION

• Resuspend in TE buffer (neutral pH + chelating agent) for DNA

• Resuspend in nuclease-free water for RNA

• Store at 5°C (refrigerator) or -70°C (ultrarefrigerator)

• AVOID -20°C — causes freeze-thaw cycles which degrade nucleic acids

• Always aliquot samples

• Use nuclease-free reagents and plasticware

POST-ISOLATION

• RNAse treatment — done after DNA isolation to remove remaining RNA

• DNAse treatment — done after RNA isolation to remove remaining DNA

• Gel Electrophoresis — quality check (visualize intact bands)

• Nanodrop Method — quantity and purity check

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