Experiment 05Enzymology & Cloning • B.Sc. / M.Sc.
Restriction Endonuclease Digestion of Plasmid DNA & Restriction Mapping
🎯 Aim: To perform single and double restriction digestion of cloning plasmid pUC19 using Type II restriction endonucleases (EcoRI, HindIII, PvuII), separate digestion products on an agarose gel, and construct a restriction cleavage map.
🔬 Theoretical Principle & Enzymatic Cleavage
Type II restriction endonucleases are homodimeric bacterial defense enzymes that recognize specific 4–8 bp palindromic sequences possessing twofold rotational symmetry and cleave phosphodiester bonds in a magnesium-dependent manner without requiring ATP hydrolysis:
- EcoRI ($5’text{-G}downarrowtext{AATTC-}3’$): Cleaves between guanine and adenine, producing 4-nucleotide single-stranded $5’$ cohesive (sticky) overhangs ($5’text{-AATT-}3’$).
- HindIII ($5’text{-A}downarrowtext{AGCTT-}3’$): Cleaves between adenine and adenine, generating $5’text{-AGCT-}3’$ cohesive termini.
- PvuII ($5’text{-CAG}downarrowtext{CTG-}3’$): Cleaves symmetrically in the center of the hexanucleotide site, generating blunt-ended DNA fragments.
- Restriction Mapping Mechanics: For a circular plasmid of size $L$:
$$text{Single-cutter enzyme: Yields exactly 1 linearized fragment of size } L$$
$$text{Double digestion: Yields } N text{ fragments whose sum equals the total circular genome size } L$$
Figure 5.1: Restriction Endonuclease Palindromic Cleavage & Plasmid Mapping
📋 Restriction Reaction Pipetting Setup (20 μL Reaction)
| Reaction Component | Undigested Control | Single Digest (EcoRI) | Single Digest (PvuII) | Double Digest (EcoRI + PvuII) |
|---|---|---|---|---|
| Sterile Nuclease-Free Water | 16.0 μL | 15.0 μL | 15.0 μL | 14.0 μL |
| 10× Universal Reaction Buffer | 2.0 μL | 2.0 μL | 2.0 μL | 2.0 μL |
| Plasmid pUC19 DNA (100 ng/μL) | 2.0 μL (200 ng) | 2.0 μL (200 ng) | 2.0 μL (200 ng) | 2.0 μL (200 ng) |
| EcoRI (10 U/μL) | — | 1.0 μL (10 U) | — | 1.0 μL (10 U) |
| PvuII (10 U/μL) | — | — | 1.0 μL (10 U) | 1.0 μL (10 U) |
Mix by gentle flicking, spin briefly, and incubate at $37^circtext{C}$ for 60 minutes. Heat-inactivate at $65^circtext{C}$ for 20 minutes before loading.
💬 Viva-Voce Questions & Model Answers
Q1: What is “Star Activity” of a restriction endonuclease and how is it prevented?
Star activity ($text{EcoRI}^*$) is the relaxation of sequence specificity where the enzyme cleaves non-canonical, degenerate sequences. It is induced by high glycerol concentration ($>5%$), non-optimal pH ($>8.0$), low ionic strength, or prolonged over-digestion. It is prevented by limiting enzyme volume to $<10%$ of total reaction volume and using recommended reaction buffers.
Q2: Why are restriction endonucleases stored in 50% glycerol at -20°C?
50% glycerol acts as a cryoprotectant that prevents water from forming ice crystals at $-20^circtext{C}$. Ice crystal formation shears and denatures the tertiary structure of sensitive enzyme dimers, causing irreversible loss of catalytic activity.
Q3: What is the difference between an Isoschizomer and a Neoschizomer?
Isoschizomers are restriction enzymes isolated from different organisms that recognize the exact same sequence and cleave at the exact same position (e.g., SphI and BbuI). Neoschizomers recognize the identical target sequence but cleave at different nucleotide positions (e.g., SmaI cleaves $5’text{-CCC}downarrowtext{GGG-}3’$ generating blunt ends, while XmaI cleaves $5’text{-C}downarrowtext{CCGGG-}3’$ generating $5’$ overhangs).