Biotechnology: Principles and Processes- Notes | Class 12 | Part 2: Tools of Recombinant DNA Technology

Tools of Recombinant DNA Technology

Restriction Enzymes (‘Molecular Scissors’)

• Enzymes that cut DNA at specific sites into fragments.
• They belong to a class of enzymes called nucleases.
• In 1963, two enzymes restricting bacteriophage growth in Escherichia coli were isolated:
  • One added methyl groups to DNA.
  • The other, a restriction endonuclease, cut DNA.
• More than 900 restriction enzymes have been isolated from over 230 bacterial strains.

Naming of the Restriction Enzymes

• The first letter indicates the genus, and the next two letters indicate the species of the prokaryotic cell from which they were isolated.
• Example: EcoRI comes from E. coli RY13, where R denotes the strain, and Roman numerals indicate the order of isolation.

Types of Restriction Enzymes

• Exonucleases: Remove nucleotides from the ends of DNA.
• Endonucleases:
  • Cut at specific positions within the DNA (e.g., EcoRI).
  • Bind to specific recognition sequences and cut both strands at specific points.
  • The first restriction endonuclease, Hind II, recognizes a specific 6-base-pair sequence, called the recognition sequence.
  • Recognize palindromic nucleotide sequences, which read the same in the 5'→3' and 3'→5' directions.

Example: Palindromic nucleotide sequence for EcoRI:

5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'

• Restriction enzymes cut strands slightly away from the palindrome’s center, between the same bases on opposite strands, creating sticky ends that form hydrogen bonds with complementary counterparts, facilitating DNA ligase action.
• When cut by the same restriction enzyme, resultant DNA fragments have identical sticky ends, joined by DNA ligases.

Cloning Vector

• A DNA molecule that carries a foreign DNA segment and replicates inside host cells (e.g., plasmids, bacteriophages).
• Plasmids: Autonomously replicating circular extra-chromosomal DNA in bacteria, with 1–2 or 15–100 copies per cell.
• Bacteriophages: Have high genome copy numbers within bacterial cells.
• Cloning vectors multiply the linked DNA to the vector’s copy number in the host.

Features Required for Cloning into a Vector

a. Origin of Replication (ori):

• Sequence where replication starts.
• DNA linked to ori replicates within host cells, controlling copy number. High-copy-number vectors are used for multiple target DNA copies.

b. Selectable Marker (Marker Gene):

• Genes that select transformants and eliminate non-transformants.
• Transformation: Introduction of DNA into a host bacterium. Transformants have the plasmid; non-transformants do not.
• In E. coli, selectable markers include antibiotic resistance genes (e.g., ampicillin, chloramphenicol, tetracycline, kanamycin). Normal E. coli lacks such resistance.

c. Cloning Sites:

• Recognition sites for restriction enzymes.
• Vectors need single or few recognition sites to link alien DNA.
• Multiple recognition sites generate several fragments, complicating cloning.
• Ligation occurs at a restriction site within an antibiotic resistance gene (e.g., in pBR322, foreign DNA is ligated at the BamHI site of the tetracycline resistance gene, forming a recombinant plasmid).



• Restriction sites: Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I.
• ori
• Antibiotic resistance genes: ampR and tetR.
• Rop: codes for the proteins involved in the replication of plasmid.

d. Vectors for Cloning Genes in Plants & Animals:

• Agrobacterium tumefaciens: Delivers T-DNA to transform plant cells into tumors, producing pathogen-required chemicals. Its tumor-inducing (Ti) plasmid is modified into a non-pathogenic cloning vector for plant gene delivery.
• Retroviruses: Transform animal cells into cancerous cells, used to deliver desirable genes into animal cells.

• Insertional Inactivation: Inserting foreign DNA into a bacterial gene inactivates it (e.g., recombinant plasmids lose tetracycline resistance).
• After introducing plasmids into E. coli, three cell types result:
  • Non-transformants: No plasmid, not resistant to tetracycline or ampicillin.
  • Transformants with non-recombinant plasmid: Resistant to both tetracycline and ampicillin.
  • Transformants with recombinant plasmid: Resistant only to ampicillin.
• Recombinants are selected by plating transformants on ampicillin medium, then transferring to tetracycline medium. Recombinants grow on ampicillin but not tetracycline; non-recombinants grow on both.
• One antibiotic resistance gene selects transformants; the inactivated gene selects recombinants.
• This selection is complex, requiring two plates. Alternative selectable markers use chromogenic substrates:
  • Recombinant DNA inserted into the β-galactosidase gene inactivates it, producing colorless colonies (recombinants).
  • Non-recombinant plasmids produce blue colonies with chromogenic substrates.

Competent Host (For Transformation with Recombinant DNA)

• Since DNA is hydrophilic, it cannot pass through cell membranes. Bacterial cells are made competent to take up alien DNA or plasmids.
• Process:
  • Treat cells with a divalent cation (e.g., calcium) to create pores in the cell wall.
  • Incubate with recombinant DNA on ice, apply heat shock at 42°C, then return to ice to enable DNA uptake.

Other Methods to Introduce Alien DNA into Host Cells

• Micro-injection: Recombinant DNA is injected directly into an animal cell’s nucleus.
• Biolistics (Gene Gun): Cells are bombarded with high-velocity micro-particles of gold or tungsten coated with DNA, suitable for plants.
• ‘Disarmed Pathogen’ Vectors: Pathogens like A. tumefaciens infect cells and transfer recombinant DNA into the host.

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Topic 1: Principles of Biotechnology Topic 3: Processes of Recombinant DNA Technology

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