Biotechnology involves the use of living organisms or their components to develop or make products for human benefit. The European Federation of Biotechnology defines it as the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services. Modern biotechnology relies heavily on genetic engineering — the ability to alter the chemistry of genetic material (DNA/RNA) and introduce it into host organisms to change their phenotype.
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Principles of Biotechnology
Two core techniques underpin modern biotechnology:
- 1. Genetic Engineering (Recombinant DNA Technology)
- The ability to alter DNA — cut it at specific locations, join pieces from different sources, and introduce the combined molecule into a host — defines genetic engineering. This relies on:
- Restriction endonucleases: molecular scissors that cut DNA at specific sequences
- DNA ligase: molecular glue that joins DNA fragments
- Vectors: DNA vehicles (plasmids, phages) to carry foreign DNA into host cells
- Host cells: cells into which the recombinant DNA is introduced (E. coli is most common)
2. Bioprocess Engineering
Maintenance of sterile (aseptic) conditions to enable growth of desired microbes or cells in large quantities to manufacture products (e.g., vaccines, enzymes, antibiotics) — using bioreactors.
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Tools of Recombinant DNA Technology
- Restriction Enzymes (Restriction Endonucleases)
- Cut double-stranded DNA at specific palindromic sequences (reads the same 5'→3' on both strands).
- Named by genus, species, strain: EcoRI comes from E. coli strain RY13. "I" = first enzyme from that organism.
- EcoRI cuts between G and AATTC in the sequence 5'-GAATTC-3', generating sticky ends (single-stranded overhangs).
- Blunt end cutters (e.g., HindII) cut exactly in the middle of the palindrome, generating no overhang.
- Sticky ends allow compatible fragments to join via complementary base pairing, then sealed by DNA ligase.
- Cloning Vectors
- Vectors carry the foreign gene into the host cell and allow its replication and expression. Requirements of a vector:
- Origin of replication (ori): sequence where DNA replication begins — ensures the vector replicates in the host
- Selectable marker: gene that helps identify transformed cells (e.g., antibiotic resistance genes like ampicillin resistance — ampR)
- Cloning sites: unique restriction enzyme sites (MCS — Multiple Cloning Site) where foreign DNA can be inserted
- Insertion of foreign DNA into a selectable marker causes insertional inactivation — a way to identify recombinants
- Common Vectors:
- pBR322: early plasmid vector with two antibiotic resistance genes (ampR and tetR). Inserting foreign DNA into tetR disrupts it — recombinants lose tetracycline resistance but retain ampicillin resistance (white/blue selection).
- BAC, YAC: Bacterial Artificial Chromosomes and Yeast Artificial Chromosomes — carry very large DNA inserts.
- phagemids, retroviruses: used in gene therapy.
- Lambda phage: head-and-tail structure; can carry foreign DNA in its non-essential central region.
- Competent Cells and Transformation
- Host cells must be made competent (able to take up foreign DNA). Methods:
- Chemical method: Treatment with divalent cations (CaCl2) increases permeability; heat shock at 42 C forces DNA in.
- Electroporation: brief electrical pulse creates temporary pores in cell membrane.
- Biolistics / Gene gun: gold or tungsten microparticles coated with DNA are blasted into plant cells.
- Microinjection: DNA is directly injected into animal cells.
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PCR (Polymerase Chain Reaction)
PCR amplifies a specific DNA segment exponentially in vitro, without need for a living organism.
- 1.Steps of PCR (each cycle = 3 steps):
- 2.Denaturation: Heat to ~94 C — hydrogen bonds break, DNA strands separate
- 3.Annealing: Cool to ~55 C — short primers (synthetic single-stranded DNA complementary to target) bind to template strands
- 4.Extension: Heat to 72 C — Taq polymerase (heat-stable enzyme from Thermus aquaticus) synthesises new DNA from the primer
After n cycles: 2n copies of the target DNA segment are produced.
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Gel Electrophoresis
- Technique to separate DNA fragments by size.
- DNA (negatively charged) migrates through agarose gel towards the positive electrode.
- Smaller fragments travel farther; larger ones travel less.
- DNA is stained with ethidium bromide (EtBr) and visualised under UV light as bright bands.
- A DNA ladder (marker with known sizes) allows estimation of fragment sizes.
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Expression of Recombinant Proteins
- For a gene to be expressed in a host:
- It must be under control of a suitable promoter (sequence that RNA polymerase recognises)
- A terminator sequence is needed downstream
- For eukaryotic genes expressed in bacteria: cDNA (complementary DNA made from mRNA using reverse transcriptase, lacking introns) is used instead of the genomic gene
- Expressed proteins are called recombinant proteins
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EcoRI cuts the sequence 5'-GAATTC-3'. Identify what type of ends it produces and explain how this helps in cloning.
EcoRI cuts between G and AATTC on both strands, leaving 4-base 5' overhangs: 5'-AATT-3' single-stranded tails (sticky ends). When another DNA fragment cut with EcoRI is mixed, complementary sticky ends pair and are sealed by DNA ligase, forming recombinant DNA efficiently.
A plasmid pBR322 has ampicillin resistance (ampR) and tetracycline resistance (tetR) genes. Foreign DNA is inserted into the BamHI site within tetR. How do you identify recombinant clones?
Plate transformed bacteria on ampicillin medium — all surviving colonies have taken up the plasmid. Replica-plate onto tetracycline medium — recombinant clones (with foreign DNA in tetR) will NOT grow. Colonies that grow on ampicillin but not tetracycline are recombinants.
Why is cDNA used instead of genomic DNA when expressing a human gene in bacteria?
Bacteria lack the machinery (spliceosomes) to remove introns from pre-mRNA. A human genomic gene contains introns. cDNA (made from mature mRNA using reverse transcriptase) lacks introns and can be directly transcribed and translated by bacterial machinery.
A researcher runs 30 cycles of PCR starting with 1 molecule of DNA. How many copies result?
After n cycles: 2n copies. After 30 cycles: 230 = approximately 109 (one billion) copies of the target DNA segment.
What properties make Taq polymerase essential for PCR?
Taq polymerase from Thermus aquaticus is a thermostable DNA polymerase — it remains active at the high denaturation temperature (~94 C) used in PCR. Ordinary DNA polymerases denature at these temperatures and would need to be added fresh each cycle.
How does gel electrophoresis separate DNA fragments, and what determines the position of a band?
DNA (negatively charged due to phosphate groups) migrates through agarose gel toward the positive electrode. The rate of migration depends on fragment size — smaller fragments migrate faster (less resistance). Position relative to a DNA ladder indicates fragment size in base pairs.
What is the role of the origin of replication (ori) in a cloning vector?
The ori sequence is the starting point for DNA replication. A vector with an ori can replicate autonomously inside the host cell, producing many copies. Without ori, the recombinant DNA would not be maintained in daughter cells after division.
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- Key Formulas / Relationships
- PCR amplification: copies after n cycles = 2n
- Sticky ends: generated by staggered cuts; blunt ends: generated by central cuts
- Insertional inactivation: insertion of foreign DNA into a resistance gene inactivates the marker
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Common mistakes
Students often confuse restriction enzymes (cut DNA) with DNA ligase (join DNA) — they have opposite roles. Remember that Taq polymerase is heat-stable, not heat-sensitive. Sticky ends are more efficient for cloning than blunt ends because complementary base pairing promotes joining. cDNA lacks introns; genomic DNA has introns.
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Summary
Recombinant DNA technology uses restriction enzymes to cut, ligase to join, vectors to carry, and host cells to replicate and express foreign genes. PCR amplifies DNA in vitro; gel electrophoresis separates DNA fragments. Together, these tools allow scientists to clone, analyse, and express any gene of interest.