Recombinant DNA Technology
Recombinant DNA (rDNA) technology involves manipulating DNA from different sources to create novel combinations. It has revolutionized medicine, agriculture, and research.
Core Tools
- Restriction Endonucleases: "Molecular scissors." Cut dsDNA at specific palindromic sequences (recognition sites, typically 4–8 bp). Create blunt ends or sticky ends (overhangs). Used to cut and paste DNA from different sources. Thousands identified; named by organism (EcoRI from E. coli, HindIII from Haemophilus influenzae).
- DNA Ligase: Joins compatible DNA fragments; seals nicks; requires ATP (or NAD+). Creates recombinant DNA molecule.
- Vectors: DNA molecules capable of autonomous replication in a host; used to carry insert DNA. Types: - Plasmids: Circular, small (1–10 kb); selectable markers (antibiotic resistance); common in bacteria - Bacteriophage (λ phage): Carries inserts up to 20 kb - BAC (Bacterial Artificial Chromosome): >100 kb; used in genome sequencing - YAC (Yeast Artificial Chromosome): Up to 1000 kb; used in Human Genome Project - Viral vectors (Retrovirus, Adenovirus, AAV): For gene therapy
- Reverse Transcriptase: Creates cDNA from mRNA (lacks introns); used to clone eukaryotic genes in bacteria.
PCR (Polymerase Chain Reaction)
Exponential amplification of specific DNA sequences in vitro. Requires: Template DNA, Two primers (flank the target), Taq polymerase (thermostable, from Thermus aquaticus), dNTPs, and thermal cycler. Three steps per cycle: (1) Denaturation (95°C — strands separate), (2) Annealing (50–60°C — primers bind), (3) Extension (72°C — Taq synthesizes new strand). Doubles DNA each cycle; 30 cycles → ~10⁹ copies. Applications: Diagnosis (COVID PCR, HIV viral load), forensics, paternity testing, prenatal diagnosis.
Cloning Strategy (General Workflow)
- Cut target gene and vector with same restriction enzyme → compatible sticky ends
- Mix and ligate → recombinant plasmid
- Transform into host bacteria (heat shock or electroporation)
- Select recombinant colonies (antibiotic resistance + blue-white screening using lacZ)
- Screen for correct insert (colony PCR, restriction digest, sequencing)
Expression Systems
- E. coli: Simple, fast, cheap; cannot glycosylate; cannot fold complex proteins correctly
- Yeast (S. cerevisiae): Can glycosylate; cheaper than mammalian; Hepatitis B vaccine
- Insect cells (Baculovirus): Good for large proteins; some glycosylation
- Mammalian cells (CHO): Full post-translational modifications; expensive; used for therapeutic antibodies, complex proteins
Therapeutic Applications
- Recombinant Proteins: Insulin, HGH, EPO, factor VIII (hemophilia), tPA (thrombolysis), G-CSF, interferons
- Monoclonal Antibodies: Trastuzumab (Herceptin), Adalimumab (Humira), Pembrolizumab (Keytruda)
- Vaccines: Hepatitis B (recombinant HBsAg), HPV (VLP), mRNA vaccines (COVID-19)
- Gene Therapy: Deliver functional gene using viral vectors; CRISPR-Cas9 gene editing
- Transgenic organisms: Golden rice (Vitamin A), knockout mice for research
DNA Sequencing
Sanger Sequencing (Chain Termination): ddNTPs (2',3'-dideoxynucleotides) terminate chain elongation; gel electrophoresis separates by size; reads ~800 bp/read. Gold standard validity.
Next-Generation Sequencing (NGS): Massively parallel; whole human genome in <24h; enables personalized medicine, cancer genomics, metagenomics.
CRISPR-Cas9: Guide RNA directs Cas9 endonuclease to specific genomic target → double strand break → precise gene editing via HR or NHEJ.