🚀 Quick Overview

Recombinant-DNA work relies on five superstar tools: restriction enzymes, DNA ligase, polymerase enzymes, cloning vectors, and a competent host cell. Together they let us cut, paste, copy, and carry any gene we want into bacteria, plants, or animals :contentReference[oaicite:21]{index=21}.

🛠️ The Main Tools

✂️ Restriction Enzymes – the molecular scissors

  • First noticed in E. coli back in 1963; Hind II was the pioneer site-specific enzyme (1968) :contentReference[oaicite:22]{index=22}.
  • They belong to the nuclease family. Exonucleases nibble from DNA ends, while endonucleases make precise internal cuts :contentReference[oaicite:23]{index=23}.
  • Every enzyme recognises a short palindromic sequence. EcoRI, for example, locks on to \(5′-\text{GAATTC}-3’\) / \(3′-\text{CTTAAG}-5’\) and cuts a little off-centre to create sticky ends that readily pair with matching overhangs :contentReference[oaicite:24]{index=24}.
  • Because sticky ends line up by base-pairing, DNA ligase can glue the pieces together, forming recombinant DNA :contentReference[oaicite:25]{index=25}.
  • After cutting, we separate fragments by agarose-gel electrophoresis. Smaller pieces zoom farther toward the anode; we visualise the orange bands under UV after ethidium-bromide staining, cut them out, and elute the DNA for cloning :contentReference[oaicite:26]{index=26}.

🧩 DNA Ligase – the molecular glue

Ligase seals the sugar-phosphate backbone wherever matching sticky ends meet. It was pivotal in the very first recombinant experiment that linked an antibiotic-resistance gene to a plasmid :contentReference[oaicite:27]{index=27}.

🔁 Polymerase Enzymes – the copiers

Once a recombinant molecule slips inside a host, the host’s own DNA polymerase (or a phage-encoded one) cranks out many identical copies, giving us gene “photocopies” on demand :contentReference[oaicite:28]{index=28}.

📦 Cloning Vectors – the delivery trucks

  • Plasmids and bacteriophages replicate independently, so an insert multiplies along with them :contentReference[oaicite:29]{index=29}.
  • Must-have features:
    • ori – the replication start, controlling copy number.
    • Selectable marker – usually antibiotic-resistance genes (AmpR, TetR, etc.) that let us spot transformants :contentReference[oaicite:30]{index=30}.
    • Unique cloning site(s) – one-of-a-kind restriction sites to avoid unwanted fragmentation. Example: BamHI site inside the TetR gene of pBR322 :contentReference[oaicite:31]{index=31}.
  • Blue-white screening: inserting DNA into the β-galactosidase gene knocks out enzyme activity; colonies stay white on chromogenic medium → easy recombinant spotting :contentReference[oaicite:32]{index=32}.
  • Plant & animal vectors: a disarmed Ti-plasmid from Agrobacterium tumefaciens ferries genes into dicot plants, while disabled retroviruses do the same in animal cells :contentReference[oaicite:33]{index=33}.

🧑‍🔬 Competent Host Cells – the willing recipients

  • Calcium-chloride treatment pokes temporary holes in the E. coli wall. A quick heat shock (0 °C → 42 °C → 0 °C) coaxes plasmids inside :contentReference[oaicite:34]{index=34}.
  • Alternative routes: micro-injection straight into an animal-cell nucleus, biolistics/gene gun that fires DNA-coated gold beads into plant cells, and disarmed pathogen vectors that infect but no longer harm the host :contentReference[oaicite:35]{index=35}.

🎯 High-Yield NEET Nuggets

  1. Name game: EcoRI = Escherichia coli strain R Y 13, first enzyme from that strain :contentReference[oaicite:36]{index=36}.
  2. Palindrome idea: identical 5’→3′ and 3’→5′ reads (GAATTC/CTTAAG) – a favourite MCQ theme :contentReference[oaicite:37]{index=37}.
  3. Vector essentials: ori, selectable marker, single cloning site – memorise pBR322 layout (AmpR, TetR) :contentReference[oaicite:38]{index=38}.
  4. Blue-white test: β-galactosidase insertional inactivation gives white recombinants – quick lab ID :contentReference[oaicite:39]{index=39}.
  5. Transformation trick: CaCl2 + heat-shock method for making E. coli competent – easy 1-mark question :contentReference[oaicite:40]{index=40}.

🌟 Happy cloning and good luck with your NEET prep! 🌟