Unit 9: Genomic DNA isolation

Plant genomic DNA isolation is the foundational step for downstream molecular work — PCR, cloning, restriction digestion, Southern blotting and library construction. The CTAB (cetyltrimethylammonium bromide) method, standardised by Murray and Thompson (1980) and refined by Doyle & Doyle (1987), is the workhorse protocol for plant tissue because it copes with the two features that break simpler kits: rigid cellulose cell walls and abundant polysaccharides and polyphenols.

I. Orientation — What DNA Isolation Requires

Any genomic DNA prep must accomplish four things in sequence: break the cell open, denature and remove protein, separate DNA from other macromolecules, and recover it in a stable, pure form. The reagents below map onto those goals.

• Target molecule: high-molecular-weight double-stranded genomic DNA (>50 kb), distinct from RNA and organellar DNA.
• Barriers unique to plants: cell wall (cellulose, lignin), large central vacuole storing hydrolytic enzymes and phenolics, and cytoplasmic polysaccharides that co-precipitate with DNA.
• Purity criteria: A₂₆₀/A₂₈₀ ratio of 1.8–2.0 (protein-free) and A₂₆₀/A₂₃₀ of >2.0 (free of polysaccharides/phenolics).
• Yield expectation: roughly 10–50 µg DNA per 100 mg fresh leaf tissue, depending on species.
• Governing chemistry: DNA is a polyanion (phosphate backbone) soluble in high-salt buffer; CTAB is a cationic detergent that forms an insoluble complex with DNA at low salt but keeps it soluble at high salt (>0.5 M NaCl). This salt-dependent behaviour is exploited throughout.

II. The CTAB Method

A. Purpose and Principle

The CTAB method lyses plant cells, complexes and removes polysaccharides and proteins, then recovers pure DNA by selective precipitation.

• Detergent action: CTAB solubilises membranes and forms complexes with polysaccharides and residual protein; at the high salt (1.4 M NaCl) of the extraction buffer, DNA–CTAB stays soluble while contaminant–CTAB complexes can be partitioned away.
• Salt-dependent precipitation: on dilution to low salt, the CTAB–DNA complex becomes insoluble and drops out, leaving many contaminants behind — the central trick that gives CTAB its selectivity.
• Two-phase clean-up: chloroform–isoamyl alcohol denatures protein and separates it into an organic/interphase layer, freeing DNA into the aqueous phase.

B. Reagents and Their Roles

Each component of the extraction buffer targets a specific contaminant or protects the DNA.

• CTAB (2% w/v): cationic detergent; lyses membranes and binds polysaccharides.
• Tris-HCl (100 mM, pH 8.0): buffer maintaining alkaline pH that keeps DNA stable and DNases inactive.
• EDTA (20 mM): chelates Mg²⁺ and Ca²⁺, the essential cofactors of DNases — stops enzymatic degradation.
• NaCl (1.4 M): the high salt that keeps DNA–CTAB soluble and helps remove polysaccharides during precipitation.
• β-mercaptoethanol (0.2%, added fresh): reducing agent that breaks disulphide bonds and neutralises oxidised polyphenols, preventing browning and DNA cross-linking.
• PVP (polyvinylpyrrolidone, 1–2%): binds polyphenols/tannins by hydrogen bonding — critical for phenolic-rich tissue.
• RNase A: hydrolyses co-purifying RNA.

Working buffer (per 100 mL):

2 g   CTAB
10 mL 1 M Tris-HCl pH 8.0   (100 mM final)
4 mL  0.5 M EDTA            (20 mM final)
28 mL 5 M NaCl              (1.4 M final)
0.2 mL β-mercaptoethanol    (add just before use)
water to 100 mL; pre-warm to 60–65 °C

C. Sample Preparation and Cell Lysis

Mechanical disruption plus warm detergent lysis opens the recalcitrant plant cell.

• Grinding: 100 mg young leaf tissue frozen in liquid nitrogen and ground to a fine powder with a chilled mortar and pestle — freezing makes the brittle wall shatter and inhibits nucleases.
• Tissue choice: young, actively growing leaves are preferred; they have thinner walls and lower secondary-metabolite load than mature tissue.
• Lysis incubation: powder mixed with ~700 µL pre-warmed CTAB buffer and incubated at 60–65 °C for 30–60 min, mixing occasionally — heat speeds membrane disruption and CTAB solubilisation while staying below the ~80 °C at which DNA denatures.

D. Protein Removal — Chloroform:Isoamyl Alcohol Extraction

Organic extraction strips protein from the lysate.

• Reagent ratio: chloroform : isoamyl alcohol = 24 : 1, added in equal volume to the lysate; chloroform denatures and precipitates protein at the interphase.
• Isoamyl alcohol role: reduces foaming and stabilises the interphase so phases separate cleanly.
• Mixing and spin: gentle inversion (never vortex — shearing fragments the long DNA), then centrifuge at ~12,000 × g for 10–15 min.
• Phase layout: three layers form —
  • Upper aqueous phase: DNA (nucleic acids, hydrophilic) — retained.
  • Interphase: denatured protein.
  • Lower organic phase: chloroform, lipids.
• Repeat: the extraction is repeated until the interphase is clear, transferring only the upper aqueous layer each time with a wide-bore tip.

E. DNA Precipitation and Recovery

Alcohol precipitation concentrates and recovers the purified DNA.

• Precipitant: 0.6–0.7 volume of chilled isopropanol (or 2 volumes ice-cold ethanol) added to the aqueous phase; alcohol dehydrates DNA and, with the salt already present, neutralises the phosphate backbone so DNA aggregates.
• Optional aid: incubation at −20 °C for 30 min improves recovery of dilute samples.
• Pelleting: centrifuge ~12,000 × g, 10 min; DNA forms a whitish pellet at the tube base.
• Ethanol wash: pellet rinsed with 70% ethanol to leach out residual salt and CTAB without redissolving the DNA (DNA is insoluble in 70% ethanol, salts are not).
• Drying and resuspension: pellet air-dried briefly (over-drying makes it hard to redissolve), then dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) or nuclease-free water for storage; TE's EDTA guards against trace DNases.
• RNA removal: RNase A (10 µg/mL, 37 °C, 30 min) digests co-precipitated RNA, which otherwise inflates A₂₆₀ readings and clutters gels.

F. Quality and Quantity Assessment

Two orthogonal checks confirm the prep worked.

• Spectrophotometry (Nanodrop/UV):
  • Concentration: A₂₆₀ of 1.0 = 50 µg/mL double-stranded DNA; e.g. A₂₆₀ = 0.4 → 20 µg/mL.
  • A₂₆₀/A₂₈₀ = 1.8–2.0: confirms freedom from protein (aromatic residues absorb at 280 nm); a ratio <1.8 signals protein carry-over.
  • A₂₆₀/A₂₃₀ > 2.0: confirms freedom from polysaccharides, phenolics and residual CTAB (which absorb near 230 nm).
• Agarose gel electrophoresis:
  • 0.8% agarose run with a DNA ladder and stained with ethidium bromide or a safe dye.
  • Intact genomic DNA: a single tight high-molecular-weight band near the well.
  • Degradation: a downward smear indicates shearing or nuclease activity.
  • RNA contamination: a low-molecular-weight cloud running ahead of the genomic band.

Worked example — yield calculation:

Given: A260 = 0.36, final volume = 100 µL, elution 1:50 dilution measured
Concentration = A260 × 50 µg/mL × dilution factor
             = 0.36 × 50 × 50 = 900 µg/mL... (if diluted)
Undiluted read: 0.36 × 50 = 18 µg/mL
Total DNA = 18 µg/mL × 0.1 mL = 1.8 µg  (scale up for larger preps)

G. Troubleshooting — Contaminants and Their Fixes

Poor plant DNA usually traces to one of three species-linked contaminants.

• Polysaccharides (viscous, gluey pellet, low A₂₆₀/A₂₃₀): raise NaCl to 2 M, add a second isopropanol precipitation, or include selective LiCl steps; the high salt keeps polysaccharides in solution while DNA precipitates.
• Polyphenols/tannins (brown pellet, oxidised DNA): increase PVP to 2–4% and β-mercaptoethanol, and keep everything cold — polyphenols bind DNA irreversibly once oxidised.
• Protein carry-over (A₂₆₀/A₂₈₀ < 1.8): repeat the chloroform:isoamyl extraction and extend proteinase/incubation.
• Sheared DNA (smear on gel): avoid vortexing and repeated freeze–thaw; use wide-bore tips and gentle inversion only.

H. Applications and Limitations

The method's balance of purity and scalability defines where it fits.

• Applications:
  • PCR and qPCR templates: CTAB DNA is clean enough for amplification, including from difficult species.
  • Molecular markers: RAPD, RFLP, AFLP, SSR and DNA barcoding all start from CTAB extracts.
  • Restriction digestion and Southern blotting: high-molecular-weight, enzyme-cleavable DNA suits these length-sensitive assays.
  • Genomic library and NGS input: provides intact template when scaled and further purified.
• Advantages:
  • Cost-effective: uses common bench reagents, no proprietary columns.
  • Handles polysaccharide/polyphenol-rich tissue better than silica-column kits, which clog.
  • Scalable from milligram to gram tissue amounts.
• Limitations:
  • Time-consuming and hazardous: chloroform and β-mercaptoethanol require a fume hood and careful disposal.
  • Species-specific tuning: salt, PVP and incubation must be re-optimised per plant.
  • Lower throughput than kits; manual phase transfers limit batch size.
  • Residual CTAB can inhibit downstream enzymes if the ethanol wash is skipped.