Biology X: Molecular Biology and Recombinant DNA Technology
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DNA and Chromosomes
DNA is a double helix (Watson & Crick, 1953) of two antiparallel strands with base pairing A-T and G-C. In eukaryotes, DNA is wound around histones into nucleosomes, which coil into chromatin and chromosomes. Genes are segments of DNA coding for RNAs or proteins.
DNA Replication
Replication is semi-conservative (Meselson & Stahl, 1958): each new double helix has one old and one new strand. Key enzymes: helicase (unwinds), primase (makes an RNA primer), DNA polymerase (adds nucleotides in the 5′ to 3′ direction), ligase (joins fragments). The leading strand is made continuously; the lagging strand is made in Okazaki fragments.
Gene Expression
The central dogma: DNA → RNA → protein.
- Transcription (nucleus): RNA polymerase binds a promoter and makes mRNA. In eukaryotes, the transcript is processed: a 5′ cap and poly-A tail are added, and introns are removed by splicing.
- Genetic code: triplet codons; 64 codons, of which 61 code for amino acids and 3 are stop codons; AUG is the start codon; the code is degenerate and nearly universal.
- Translation (ribosome): mRNA is read; tRNA brings amino acids (anticodon pairs with codon); peptide bonds form; the chain ends at a stop codon.
- Regulation: the lac operon in E. coli (Jacob & Monod, 1961) shows how genes are switched on and off in response to lactose. Eukaryotes regulate expression by transcription factors, chromatin changes, and RNA processing.
Mutations
A mutation is a change in DNA sequence. Point mutations: silent, missense, nonsense; frameshift mutations (insertions or deletions that are not multiples of three) usually have severe effects; chromosomal mutations (deletion, duplication, inversion, translocation). Mutagens include radiation and chemicals. Mutations provide the raw material of evolution and cause diseases such as sickle cell anaemia (a single base change in the β-globin gene).
Recombinant DNA Technology
| Technique | Principle | Use |
|---|---|---|
| Restriction enzymes | Cut DNA at specific sequences (for example EcoRI) | Making DNA fragments |
| DNA ligase | Joins DNA fragments | Making recombinant DNA |
| Vectors (plasmids, viruses) | Carry genes into host cells | Cloning and gene transfer |
| PCR (polymerase chain reaction) | Cycles of denaturation (about 94 to 95 °C), annealing (about 50 to 65 °C), and extension (about 72 °C) with Taq polymerase amplify DNA | Diagnosis, forensics, research |
| Gel electrophoresis | DNA fragments move through a gel in an electric field; shorter fragments move farther | Separating DNA |
| DNA sequencing | Reading base order (Sanger and next-generation methods) | Genomics, diagnosis |
| DNA profiling | Compares short tandem repeats | Forensic science, paternity |
| CRISPR-Cas9 | A guide RNA directs the Cas9 enzyme to cut a target sequence (Jinek et al., 2012) | Gene editing |
Applications and Ethics
- Medicine: recombinant insulin, growth hormone, vaccines, gene therapy, diagnostics, and personalized medicine.
- Agriculture: genetically modified crops (insect-resistant Bt crops, herbicide-tolerant crops, biofortified crops).
- Industry and environment: enzymes, bioremediation, biofuels.
- Biosafety: the Cartagena Protocol on Biosafety (Convention on Biological Diversity, 2000) regulates the transboundary movement of living modified organisms. National frameworks govern research and release.
- Ethical questions: safety, consent, equity, patents, genetic privacy, human germline editing, and effects on biodiversity. Evaluate benefits and risks with evidence and respect for different views.
Human Genetic Disorders
Sickle cell disease (autosomal recessive), cystic fibrosis, thalassaemia (relevant in Sri Lanka and South Asia), Down syndrome, Turner and Klinefelter syndromes, haemophilia. Genetic counselling and screening help families make informed decisions.
Common Mistakes
- Mixing up transcription and translation.
- Forgetting that mRNA is complementary to the template strand.
- Describing PCR steps in the wrong temperature order.
CHAPTER 13