4.4 Molecular Genetics and Biotechnology
Key Takeaways
- Point mutations include silent (no amino acid change), missense (one amino acid swap), and nonsense (premature stop) changes; frameshift mutations from insertions or deletions not divisible by three scramble every downstream codon.
- The lac operon in E. coli is an inducible system turned ON by lactose (the inducer relieves repression), while the trp operon is a repressible system turned OFF when tryptophan is abundant.
- Eukaryotic gene regulation relies on combinations of transcription factors binding enhancers and silencers, often far from the gene, looped to the promoter by mediator proteins.
- PCR amplifies a target DNA sequence through repeated cycles of denaturation at about 95 C, primer annealing near 55 C, and extension at about 72 C using a heat-stable DNA polymerase such as Taq.
- CRISPR-Cas9 uses a guide RNA to direct the Cas9 nuclease to a specific genomic sequence, where it cuts both DNA strands and the cell's repair machinery introduces the desired edit.
Why This Section Is High-Yield
The Praxis Biology test will give you a short scenario - a mutation, an operon diagram, a PCR protocol, or a gel image - and ask you to predict the outcome. The trick is to keep three vocabularies straight: the kinds of mutations, the regulation logic, and the lab tools.
Mutations
Point Mutations
A point mutation changes a single nucleotide.
| Type | What Changes | Effect on Protein |
|---|---|---|
| Silent | One codon to a synonymous codon (often at the third/wobble position) | None - same amino acid |
| Missense | One codon to a codon for a different amino acid | One amino acid substituted; effect ranges from negligible to severe (sickle cell anemia: GAG -> GUG at codon 6 of beta-globin) |
| Nonsense | Sense codon -> stop codon | Premature termination; usually severe |
Frameshift Mutations
Insertion or deletion of nucleotides not in multiples of three shifts the reading frame, so every codon downstream is changed. These are almost always severe because they typically introduce a premature stop codon as well.
Large-Scale Mutations
- Deletion: loss of a chromosomal segment.
- Duplication: a segment is repeated.
- Inversion: a segment flips end-to-end within the same chromosome.
- Translocation: a segment moves to a non-homologous chromosome (e.g., the BCR-ABL fusion in chronic myeloid leukemia caused by the t(9;22) Philadelphia chromosome).
Gene Regulation in Prokaryotes: Operons
Prokaryotes group functionally related genes under one promoter as an operon. Two operons dominate the curriculum.
The lac Operon (Inducible)
- Genes: lacZ (beta-galactosidase), lacY (permease), lacA (transacetylase).
- The lac repressor (lacI) is normally bound to the operator and blocks transcription.
- When lactose is present, allolactose (the inducer) binds the repressor, changes its shape, and removes it from the operator -> transcription ON.
- Glucose presence reduces cAMP, lowers CAP activity, and dampens expression even with lactose - the basis of catabolite repression.
The trp Operon (Repressible)
- Genes for tryptophan biosynthesis.
- The trp repressor is normally INACTIVE.
- When tryptophan is abundant, tryptophan binds the repressor as a corepressor, activates it, and the repressor binds the operator -> transcription OFF.
Mnemonic: Inducible = OFF by default, turned ON by the substrate. Repressible = ON by default, turned OFF when the end product accumulates.
Gene Regulation in Eukaryotes
Eukaryotes use combinatorial control:
- General transcription factors assemble at the promoter to recruit RNA polymerase II.
- Specific transcription factors bind enhancers (positive regulators) or silencers (negative regulators), often thousands of base pairs from the gene.
- DNA loops bring distant enhancers physically close to the promoter via mediator complexes.
- Chromatin remodeling, histone acetylation (open chromatin), and DNA methylation (typically silencing) provide an extra epigenetic layer.
Core Biotechnology Tools
Polymerase Chain Reaction (PCR)
PCR amplifies a target sequence from minute amounts of DNA. Each cycle has three temperature-controlled steps:
| Step | Temperature | What Happens |
|---|---|---|
| Denaturation | ~95 C | Double-stranded DNA melts into single strands |
| Annealing | ~55 C (varies with primer Tm) | Primers bind their complementary target sequences |
| Extension | ~72 C | Taq polymerase extends from the primers, building new strands |
After n cycles, the target is amplified roughly 2^n - so 30 cycles can yield ~ a billion copies. Taq comes from Thermus aquaticus and is heat-stable so it survives the 95 C step.
Gel Electrophoresis
Negatively charged DNA fragments migrate through an agarose gel toward the positive electrode. Smaller fragments move faster, so the gel sorts DNA by size. Fragments are visualized with a stain (such as ethidium bromide or SYBR Green) and compared with a size ladder.
Restriction Enzymes and Recombinant DNA
Restriction enzymes are bacterial nucleases that cut DNA at specific palindromic sequences, often producing complementary sticky ends. EcoRI cuts at GAATTC. To make recombinant DNA, the same enzyme is used to cut both the plasmid vector and the gene of interest; the matching sticky ends anneal and DNA ligase seals the backbone.
Transformation
Bacteria are made competent (often with calcium chloride and a brief heat shock) so they take up the recombinant plasmid. Transformed bacteria carrying the plasmid's selection marker (e.g., ampicillin resistance) survive on selective media.
CRISPR-Cas9
CRISPR-Cas9 is a genome-editing system adapted from a bacterial adaptive immune defense.
- A short single guide RNA (sgRNA) carries a 20-nucleotide sequence complementary to the target.
- The Cas9 nuclease uses the sgRNA to find and bind the target (a PAM sequence such as NGG must sit adjacent on the genome).
- Cas9 creates a double-strand break, which the cell repairs by non-homologous end joining (NHEJ) - typically inserts/deletes that knock out the gene - or by homology-directed repair (HDR) when a donor template is supplied, enabling precise edits.
CRISPR is faster and cheaper than older tools (zinc finger nucleases, TALENs) and has applications from sickle cell disease therapy to crop engineering.
An E. coli strain has a mutation in the lacI gene that produces a non-functional lac repressor. The bacteria are grown in a medium containing glucose but no lactose. Which of the following is expected?
A researcher runs a PCR reaction with the following thermocycler settings: 95 C, then 55 C, then 72 C, repeated 30 times. Which enzyme is essential for the reaction to work, and why?