Unit 3: Restriction digestion of DNA

Restriction digestion is the controlled, sequence-specific cleavage of double-stranded DNA using bacterial enzymes, and it forms the analytical backbone of gene cloning, mapping and fingerprinting. This unit deals with cutting plant genomic DNA with restriction endonucleases and then measuring the resulting fragment sizes by agarose gel electrophoresis.

I. Orientation: Restriction Endonucleases and the Digestion Principle

Restriction enzymes were discovered in the late 1960s–70s (Arber, Smith and Nathans, Nobel Prize 1978) as part of the bacterial defence against invading phage DNA. They recognise specific short sequences and cleave the phosphodiester backbone, giving reproducible fragments that can be sized on a gel.

• Nomenclature: Named from the source organism — EcoRI from Escherichia coli strain RY13; the first letter is genus, next two the species, then strain and Roman numeral for order of discovery.
• Recognition site: Usually a 4–8 bp palindrome, e.g. EcoRI recognises 5'-GAATTC-3'; the complementary strand reads identically 5'→3'.
• Cut geometry — two outcomes:
  • Sticky (cohesive) ends: staggered cut leaving single-stranded overhangs, e.g. EcoRI cuts between G and A.
  • Blunt ends: cut at the axis of symmetry leaving no overhang, e.g. SmaI on CCCGGG.
• Cofactor requirement: Type II enzymes need Mg²⁺; they cut at or near the recognition site and are the workhorses of the laboratory.
• Frequency of cutting: For random DNA, a recognition site of n bases occurs on average once every 4ⁿ base pairs (a 6-cutter ≈ every 4096 bp).

II. Restriction Digestion of Plant DNA

A. Purpose and Principle

The aim is to expose purified plant genomic DNA to a chosen endonuclease under optimal buffer conditions so that every recognition site is cleaved, producing a defined population of fragments.

• Principle: Enzyme scans the DNA, binds its recognition site, and hydrolyses both strands; the number and size of fragments depend on how often the site appears in the genome.
• Why plant DNA is a special case: Plant tissue carries thick cell walls, high polysaccharide and polyphenolic content, and abundant secondary metabolites that co-purify and can inhibit enzymes.

B. Restriction Digestion of Plant DNA — Template Preparation and Quality

Clean, high-molecular-weight template is the single biggest determinant of a successful digest.

• Extraction: CTAB (cetyltrimethylammonium bromide) method is standard for plants; CTAB complexes polysaccharides and separates them from nucleic acid at high salt.
• Inhibitor removal:
  • Polyphenols/tannins: added PVP (polyvinylpyrrolidone) binds them.
  • RNA: removed with RNase A so it does not obscure small fragments on the gel.
• Purity check: spectrophotometric ratios — A₂₆₀/A₂₈₀ ≈ 1.8 (protein-free) and A₂₆₀/A₂₃₀ ≈ 2.0 (free of polysaccharide/phenolic carryover).
• Integrity: undegraded genomic DNA runs as a tight high-molecular-weight band; a smear indicates shearing that will confound digestion patterns.

C. Setting Up the Digestion Reaction

The reaction is assembled from defined components in the enzyme's optimal buffer.

• Standard 20 µL reaction:
  
  TEXT

    Genomic DNA (0.5–1 µg)        x µL
    10X restriction buffer         2 µL
    Restriction enzyme (10 U/µg)   1 µL  (add last)
    Nuclease-free water        to 20 µL

• Component roles:
  • 10X buffer: supplies Mg²⁺, sets pH (Tris, ~7.5) and ionic strength (NaCl/KCl) matched to the enzyme.
  • Unit definition: 1 unit (U) = enzyme needed to digest 1 µg of substrate DNA in 1 hour at optimal temperature.
• Incubation: typically 37 °C for 1–4 h; enzyme is added last and the tube mixed gently to avoid shearing.
• Enzyme fraction limit: enzyme volume kept < 10 % of total reaction because storage glycerol (~50 %) above this level causes non-specific cutting.

D. Star Activity and Reaction Fidelity

Under non-ideal conditions enzymes cut relaxed, non-canonical sites — "star activity" — corrupting the pattern.

• Triggers: excess glycerol (>5 %), high enzyme units, low ionic strength, high pH, or Mn²⁺ substituting for Mg²⁺.
• Control: keep glycerol low, use recommended units, incubate for a defined time, and stop the reaction on time.
• Stopping the reaction: heat inactivation (e.g. 65 °C for 20 min) or adding EDTA, which chelates Mg²⁺ and halts catalysis.

E. Digestion Modes: Single, Double and Partial

Different mapping needs call for different digestion strategies.

1. Complete digestion (single/double): enzyme(s) allowed to cut every site to completion.
   • Double digest: two enzymes in one tube require a compatible common buffer; otherwise digest sequentially with a clean-up in between.
2. Partial digestion: limited time or reduced enzyme so only some sites cut, yielding overlapping fragments used to order sites along a large clone.

F. Applications and Limitations

• Applications: construction of restriction maps, RFLP-based genetic diversity analysis in crops, preparing inserts and vectors for cloning, and confirming recombinant constructs.
• Limitations: plant inhibitors reduce efficiency; CpG/cytosine methylation in plant genomes can block methylation-sensitive enzymes (e.g. some sites resistant to HpaII but cut by isoschizomer MspI).

III. Size Determination by Agarose Gel Electrophoresis

A. Purpose and Principle

Electrophoresis separates the digested fragments by size so their lengths can be estimated against a known ladder.

• Principle: DNA carries a uniform negative charge from its phosphate backbone, so in an electric field it migrates from the cathode (–) toward the anode (+).
• Sieving: the agarose gel is a porous matrix; small fragments thread through pores faster than large ones, so migration distance is inversely related to fragment size.
• Governing relationship:
  
  TEXT

    log₁₀(fragment size in bp)  ∝  – (migration distance)

  
  Over the resolving range, log of size falls linearly with distance travelled.

B. Gel Preparation and Apparatus

The matrix concentration is chosen to match the fragment size range under study.

• Agarose concentration: higher % = smaller pores = better resolution of small fragments.
  • 0.8 % resolves ~0.5–10 kb; 2 % resolves ~0.1–2 kb.
• Buffer: TAE (Tris-acetate-EDTA) or TBE (Tris-borate-EDTA) both conduct current and hold pH; TBE gives sharper bands for small fragments, TAE better for large ones.
• Setup: molten agarose poured with a comb to form loading wells; solidified gel submerged in the same buffer (submarine electrophoresis).

C. Loading, Running and Visualisation

Samples are prepared with tracking dye, run at controlled voltage, then stained for viewing.

• Loading (gel-loading) dye:
  • Glycerol/sucrose: density sinks the sample into the well.
  • Bromophenol blue / xylene cyanol: visible fronts marking migration progress.
• Voltage: typically 5 V/cm; excessive voltage overheats the gel and smears bands.
• Staining:
  • Ethidium bromide (EtBr): intercalates between base pairs and fluoresces orange under UV (~300 nm); it is a mutagen and handled with care.
  • Safer alternatives: SYBR-type dyes.
• Documentation: bands photographed on a UV transilluminator or gel-doc system.

D. Size Determination by Agarose Gel Electrophoresis — Using a Standard Ladder

Fragment size is read off by comparing sample bands to a co-run DNA size marker.

• DNA ladder: a mixture of fragments of known sizes (e.g. a 1 kb ladder, or λ DNA cut with HindIII giving fragments 23130, 9416, 6557, 4361, 2322, 2027, 564, 125 bp).
• Standard curve method:
  • Measure the migration distance of each ladder band from the well.
  • Plot log₁₀(size) on the y-axis against distance on the x-axis — a near-straight line.
  • Read the unknown fragment's distance against the line to interpolate its size.
• Worked example:
  • A ladder gives 10 kb at 12 mm and 1 kb at 42 mm; log-size falls from 4.0 to 3.0 over 30 mm.
  • An unknown band migrates 27 mm → interpolated log-size = 4.0 − (15/30 × 1.0) = 3.5 → ≈ 3.16 kb.
• Genome-size caveat: a plant genome cut with a 6-cutter yields thousands of fragments that overlap into a smear, so discrete sizing works for cloned DNA or plasmids, while genomic RFLP requires Southern blotting to reveal individual bands.

E. Factors Affecting Migration and Accuracy

Several variables besides length alter how far a fragment travels.

• Fragment conformation: for plasmids, supercoiled, linear and nicked forms migrate differently even at the same size; only linearised DNA sizes reliably.
• Agarose %, voltage, and buffer ionic strength: each shifts band positions, so ladder and samples must run on the same gel under identical conditions.
• Resolution limit: very large fragments co-migrate near the well; separating them needs pulsed-field gel electrophoresis (PFGE).
• Overloading: too much DNA per well distorts bands and inflates apparent size.

F. Significance in Plant Biotechnology

• Verification: confirms that a digest worked and that fragment sizes match the predicted map.
• Diversity studies: banding patterns from digested plant DNA underpin RFLP markers for cultivar identification and linkage mapping.
• Downstream steps: correctly sized fragments are excised and gel-purified for ligation, sequencing or probe preparation.