Unit 1: Genomic DNA from plant

BTY555 — Biotechnology Laboratory-I 7 min read

Plant genomic DNA isolation is the foundational technique on which most downstream molecular work in this laboratory depends. This unit establishes what genomic DNA is, why plant tissue is uniquely difficult to work with, and how the CTAB (cetyltrimethylammonium bromide) method overcomes those difficulties to yield high-molecular-weight DNA suitable for PCR, restriction digestion, cloning, and Southern blotting.

I. Orientation: Genomic DNA and the Plant Cell Problem

Genomic DNA is the complete complement of double-stranded DNA carried in the nucleus (and, in plants, additionally in chloroplasts and mitochondria) of every cell. Extraction means lysing cells, separating DNA from proteins, RNA, lipids and secondary metabolites, and recovering it in pure, intact form (typically 20–50 kb fragments).

Target molecule: high-molecular-weight double-stranded DNA; measured in nanograms to micrograms, quantified spectrophotometrically at 260 nm.
Purity benchmarks: A₂₆₀/A₂₈₀ ratio of 1.8–2.0 indicates protein-free DNA; A₂₆₀/A₂₃₀ of ~2.0–2.2 indicates freedom from polysaccharides, phenolics and CTAB.
Why plants are hard:
- Cell wall: a rigid cellulose–lignin barrier absent in animal cells, requiring mechanical grinding to breach.
- Polysaccharides: abundant carbohydrates co-precipitate with DNA, producing a viscous, glassy pellet that inhibits Taq polymerase and restriction enzymes.
- Polyphenols and tannins: oxidise to quinones that bind covalently to DNA, browning the extract and rendering DNA unamplifiable.
- Secondary metabolites: alkaloids, latex and pigments vary by species and interfere unpredictably.
Governing principle of CTAB: a cationic detergent that forms an insoluble complex with nucleic acids at low salt but keeps them soluble at high salt (>0.7 M NaCl), while polysaccharides and other contaminants partition away — this differential solubility is the whole basis of the method.

II. Extraction of Genomic DNA from Plant by Using CTAB Method

The CTAB method (developed by Murray and Thompson, 1980; popularised by Doyle and Doyle, 1987) isolates DNA by detergent-mediated lysis, selective complex formation, organic extraction of proteins, and alcohol precipitation of nucleic acids. Its purpose is to deliver clean, high-yield genomic DNA even from tissue rich in polysaccharides and phenolics.

A. Reagents of the CTAB Extraction Buffer

Each buffer component performs one defined chemical job; understanding them explains every later step.

CTAB (2% w/v): the cationic detergent that solubilises membrane lipids and proteins and forms the CTAB–nucleic-acid complex.
Tris-HCl (100 mM, pH 8.0): buffers the extract at slightly alkaline pH to keep DNA stable and DNases minimally active.
EDTA (20 mM): chelates Mg²⁺ and Ca²⁺, the essential cofactors of DNases, thereby inhibiting DNA degradation.
NaCl (1.4 M): the high-salt condition that keeps DNA in solution while removing polysaccharides; salt shields DNA phosphate backbone from CTAB until dilution.
β-mercaptoethanol (0.2% v/v, added fresh): a reducing agent that prevents phenolic oxidation by breaking disulfide bonds and denaturing oxidase enzymes, avoiding the brown quinone–DNA complexes.
PVP – polyvinylpyrrolidone (1–2%, optional): hydrogen-bonds to polyphenols and sequesters them away from DNA, valuable for tannin-rich tissue.

TEXT

CTAB extraction buffer (per 100 mL)
  2 g      CTAB
  10 mL    1 M Tris-HCl, pH 8.0
  4 mL     0.5 M EDTA, pH 8.0
  28 mL    5 M NaCl
  0.2 mL   β-mercaptoethanol (just before use)
  water to 100 mL

B. Tissue Homogenisation and Cell Lysis

The step converts intact tissue into a suspension in which membranes are dissolved and DNA is released.

Sample: 100–200 mg fresh young leaf (meristematic tissue has fewer secondary metabolites and more nuclei per gram).
Grinding: tissue is frozen in liquid nitrogen (−196 °C) and ground to a fine powder with mortar and pestle; freezing makes the brittle cell wall shatter and inactivates DNases.
Lysis: the powder is mixed with pre-warmed CTAB buffer and incubated at 60–65 °C for 30–60 min, with gentle inversion.
- Heat role: raises detergent action, denatures proteins including nucleases, and speeds membrane disruption without shearing DNA.
- Outcome: nuclear and organellar membranes lyse, releasing genomic DNA into a CTAB-rich solution.

C. Chloroform–Isoamyl Alcohol Deproteinisation

The step strips proteins and lipids from the aqueous DNA phase by liquid–liquid extraction.

Reagent: chloroform : isoamyl alcohol (24:1) added in equal volume to the lysate and mixed by gentle inversion to form an emulsion.
- Chloroform: denatures and precipitates proteins at the interface and dissolves lipids and pigments into the organic layer.
- Isoamyl alcohol: reduces foaming and stabilises the interphase, giving a sharp boundary.
Centrifugation: ~12,000 × g for 10 min separates three layers:
1. Upper aqueous phase: contains the DNA (CTAB–DNA complex still soluble in high salt).
2. Interphase: a white film of denatured protein.
3. Lower organic phase: chloroform with dissolved lipids and pigments.
Recovery: the upper aqueous phase is pipetted off carefully without disturbing the interphase; the extraction may be repeated until the interphase is clean.

D. Precipitation of Genomic DNA

The step recovers DNA from solution as a visible precipitate by removing the shielding salt and dehydrating the nucleic acid.

Precipitant: chilled isopropanol (0.6–0.7 volume) or absolute ethanol (2 volumes) added to the aqueous phase.
- Mechanism: alcohol displaces water from the DNA hydration shell; at reduced salt the negatively charged phosphate backbone aggregates and DNA precipitates.
- Isopropanol vs ethanol:
  1. Isopropanol: needs less volume and precipitates DNA faster, but also co-precipitates more salt — chosen when tube volume is limiting.
  2. Ethanol: needs 2 volumes but gives cleaner precipitate with less salt carry-over.
Incubation: −20 °C for 30 min to 1 h improves yield for dilute samples.
Pelleting: centrifugation at ~12,000 × g for 10 min collects DNA as a whitish pellet; threadlike DNA may be spooled out with a hooked pipette.

E. Washing and Resuspension

The step removes residual salt, CTAB and alcohol, then redissolves purified DNA for storage.

Wash: the pellet is rinsed with 70% ethanol, which dissolves and carries away salts and CTAB while DNA, being insoluble in aqueous ethanol, stays as a pellet.
Drying: the pellet is air-dried (or briefly vacuum-dried) to evaporate ethanol; over-drying makes DNA hard to resuspend, so drying stops while a faint film of moisture remains.
Resuspension: DNA is dissolved in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) or nuclease-free water.
- TE role: Tris buffers pH; EDTA chelates metal ions to guard against residual nuclease activity during storage.
RNase treatment: RNase A (10–50 µg/mL, 37 °C, 30 min) is added to digest co-purified RNA, since RNA otherwise inflates A₂₆₀ readings and clutters gel lanes.

F. Quality Assessment of Extracted DNA

The step confirms that the isolated DNA is intact, pure and correctly quantified before downstream use.

Spectrophotometry (UV, 260/280/230 nm):
- Concentration: for dsDNA, an A₂₆₀ of 1.0 ≈ 50 µg/mL.