Cell Biology: Molecular biology, gene expression and laboratory techniques

🔬 Molecular biology, gene expression and laboratory techniques. Molecular biology - how DNA is copied, repaired and expressed - underpins genetic diagnosis, cancer biology, antiviral and antibiotic therapy, vaccines and modern laboratory medicine. Clinicians now order genomic tests routinely (the NHS Genomic Medicine Service), interpret PCR results daily, and use mRNA vaccines and gene therapies. This chapter covers DNA structure and replication, DNA repair, transcription, translation and gene regulation, and the laboratory techniques you need to understand. Inheritance patterns and clinical genetics are in the Clinical Genetics chapters.
Your four questions: Where in the flow of genetic information is the defect or drug acting (replication, repair, transcription, translation)? What type of mutation is it? Which test detects it (and what can it miss)? How is the result used clinically?

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DNA Structure and Replication

• DNA - double helix of antiparallel strands; sugar–phosphate backbone; bases adenine–thymine (2 hydrogen bonds) and guanine–cytosine (3 hydrogen bonds - GC-rich DNA is more stable). Purines: A, G; pyrimidines: C, T (U in RNA).
• Human genome - about 3.1 billion base pairs, about 20,000 protein-coding genes (only about 1–2% of the genome is coding); 23 pairs of chromosomes; mitochondrial DNA separately.
• Chromatin - DNA wound around histone octamers (nucleosomes); euchromatin (open, active) and heterochromatin (condensed, silent).
• Replication (S phase) - semi-conservative; helicase unwinds, primase lays RNA primers, DNA polymerase synthesises 5'→3' (leading strand continuously, lagging strand in Okazaki fragments joined by ligase), topoisomerases relieve supercoiling.
• Drug targets: topoisomerase I (irinotecan, topotecan), topoisomerase II (etoposide, anthracyclines); bacterial DNA gyrase / topoisomerase IV (fluoroquinolones); viral DNA polymerase (aciclovir - requires viral thymidine kinase for activation); reverse transcriptase (HIV - NRTIs, NNRTIs).

DNA Damage and Repair

Repair pathway	Damage repaired	Defect causes
Mismatch repair (MLH1, MSH2, MSH6, PMS2)	Replication errors (mismatches)	Lynch syndrome (HNPCC) - colorectal (right-sided), endometrial, ovarian, gastric, urinary tract cancers; tumours show microsatellite instability (MSI) - all colorectal and endometrial cancers are tested; MSI-high tumours respond to immunotherapy
Nucleotide excision repair	Bulky lesions - UV-induced thymine dimers	Xeroderma pigmentosum - extreme sun sensitivity, skin cancers in childhood
Base excision repair	Single damaged bases (oxidation, deamination)	MUTYH-associated polyposis (autosomal recessive)
Homologous recombination (BRCA1, BRCA2, PALB2)	Double-strand breaks (accurate)	Hereditary breast and ovarian cancer (also prostate, pancreatic); PARP inhibitors (olaparib) exploit the defect - synthetic lethality
Non-homologous end joining	Double-strand breaks (error-prone)	Some severe combined immunodeficiencies (V(D)J recombination defects)
Fanconi anaemia pathway	Interstrand cross-links	Fanconi anaemia - bone marrow failure, radial ray defects, short stature, cancer
ATM	Double-strand break signalling	Ataxia telangiectasia - cerebellar ataxia, telangiectasia, immunodeficiency, lymphoma, radiation sensitivity

• DNA-damaging agents: UV light, ionising radiation, smoking (polycyclic hydrocarbons), aflatoxin (TP53 mutation, liver cancer), alkylating chemotherapy and platinum compounds (cross-links).

Types of Mutation

Type	Effect	Example
Silent (synonymous)	Same amino acid	Usually harmless
Missense	Different amino acid	Sickle cell (glutamate → valine in β-globin)
Nonsense	Premature stop codon → truncated protein	Some Duchenne muscular dystrophy, β-thalassaemia
Frameshift	Insertion or deletion not a multiple of 3 → reading frame shifted	Duchenne (out-of-frame deletion) versus Becker (in-frame deletion - milder, partially functional dystrophin)
In-frame deletion	Loss of amino acids	Cystic fibrosis F508del (loss of phenylalanine)
Splice-site	Abnormal mRNA splicing	Some β-thalassaemias, spinal muscular atrophy (SMN2 splicing - nusinersen modifies it)
Trinucleotide repeat expansion	Variable; may show anticipation (earlier, more severe in successive generations)	Huntington's (CAG), myotonic dystrophy (CTG), fragile X (CGG), Friedreich's ataxia (GAA - recessive), spinocerebellar ataxias
Copy number variants	Deletion/duplication of segments	22q11.2 deletion (DiGeorge), CMT1A (PMP22 duplication)

• Germline (inherited, in all cells) versus somatic (acquired, in tumours) mutations; mosaicism - two cell lines in one person.
• Variant classification (ACMG): pathogenic, likely pathogenic, variant of uncertain significance (VUS - do not act clinically), likely benign, benign.

Transcription and RNA Processing

• Transcription - RNA polymerase II makes messenger RNA from the DNA template, starting at promoters (TATA box) with transcription factors; enhancers and silencers regulate from a distance.
• mRNA processing (eukaryotes): 5' cap, 3' poly-A tail, splicing (removal of introns by the spliceosome - antibodies to snRNPs (anti-Smith) in SLE). Alternative splicing lets one gene make several proteins.
• Other RNAs: rRNA (ribosomes), tRNA (amino acid delivery), microRNAs (silence mRNA), long non-coding RNAs.
• Drugs and toxins: rifampicin (bacterial RNA polymerase), actinomycin D (DNA intercalation), α-amanitin (death cap mushroom - RNA polymerase II → liver failure).

Translation and Post-translational Modification

• Genetic code - triplet codons, degenerate (several codons per amino acid), nearly universal; AUG start (methionine); stop codons UAA, UAG, UGA.
• Translation on ribosomes: initiation, elongation, termination.
• Antibiotics targeting bacterial (70S) ribosomes: 30S - aminoglycosides (bactericidal - misreading), tetracyclines (block tRNA binding); 50S - macrolides, clindamycin, chloramphenicol, linezolid (blocks initiation complex), fusidic acid (EF-G).
• Diphtheria and Pseudomonas exotoxin A inactivate elongation factor 2 (EF-2) in human cells; Shiga toxin and ricin inactivate the 60S ribosome.
• Post-translational modification: phosphorylation (signalling), glycosylation (Golgi), γ-carboxylation (vitamin K-dependent clotting factors - warfarin), hydroxylation of proline and lysine (collagen - vitamin C), ubiquitination (proteasomal degradation), cleavage of prohormones (proinsulin → insulin + C-peptide), lipidation.

Gene Regulation and Epigenetics

• Epigenetics - heritable changes in gene expression without changes in DNA sequence: DNA methylation (CpG islands - silencing), histone modification (acetylation opens chromatin; deacetylation closes it), non-coding RNAs.
• Genomic imprinting - expression depends on the parent of origin: Prader–Willi syndrome (loss of paternal 15q11–13 - hyperphagia, obesity, hypotonia, hypogonadism, learning difficulty) and Angelman syndrome (loss of maternal UBE3A - severe learning disability, seizures, ataxia, happy demeanour); Beckwith–Wiedemann syndrome (11p15).
• X-inactivation (lyonisation) - one X is randomly inactivated in each female cell → variable expression in carrier females of X-linked conditions (e.g. symptomatic DMD carriers).
• Epigenetic drugs - azacitidine and decitabine (hypomethylating agents - myelodysplasia, AML), HDAC inhibitors.
• Environmental epigenetics - maternal nutrition, smoking and stress affect offspring gene expression (developmental origins of disease).

PCR and Its Variants

• Polymerase chain reaction - exponential amplification of a target DNA sequence using primers, heat-stable (Taq) polymerase and repeated cycles of denaturation (about 95°C), annealing (about 55–65°C) and extension (72°C); about 30–40 cycles.
• RT-PCR (reverse transcription PCR) - converts RNA to cDNA first; detects RNA viruses (SARS-CoV-2, influenza, HIV viral load, HCV RNA) and gene fusion transcripts (BCR-ABL monitoring in CML).
• Quantitative (real-time) PCR - measures amplification as it happens; the cycle threshold (Ct) is inversely related to the amount of starting target (low Ct = high viral load).
• Multiplex PCR - many targets at once (respiratory and GI pathogen panels, meningitis panels).
• Strengths: very sensitive and specific, fast. Limitations: detects nucleic acid, not live organisms (positive after recovery); contamination causes false positives; sequence changes at primer sites cause false negatives; does not give antibiotic sensitivities (except known resistance genes - e.g. mecA for MRSA, rifampicin resistance in TB with GeneXpert).

Sequencing and Genomic Testing

• Sanger sequencing - reads one fragment (up to about 1,000 bases) accurately; confirms specific variants and cascade testing in families.
• Next-generation sequencing (NGS) - massively parallel: gene panels (targeted - e.g. hereditary cancer panels, cardiomyopathy panels), whole exome sequencing, whole genome sequencing (NHS England offers WGS for rare disease in children and some cancers through the Genomic Medicine Service and the National Genomic Test Directory).
• Tumour (somatic) testing - identifies targetable mutations (EGFR, ALK, BRAF, KRAS, HER2, BRCA, MSI); liquid biopsy - circulating tumour DNA from blood.
• Pathogen sequencing - outbreak tracing (whole genome sequencing of TB, MRSA, SARS-CoV-2 variants), resistance prediction.
• Non-invasive prenatal testing (NIPT) - cell-free fetal DNA in maternal blood for trisomies (high sensitivity, but positives need confirmation by invasive testing).
• Incidental findings and consent - genomic tests may reveal unexpected results (other disease risks, non-paternity); pre-test counselling is essential.

Cytogenetics - Karyotype, FISH and Microarray

• Karyotype - chromosomes in metaphase, stained (G-banding); detects aneuploidy and large structural changes (>5–10 Mb): trisomies, translocations (Philadelphia chromosome), large deletions.
• FISH (fluorescence in situ hybridisation) - fluorescent probes for specific sequences; detects microdeletions (22q11.2), gene amplification (HER2 in breast cancer), translocations (BCR-ABL); rapid prenatal aneuploidy testing.
• Chromosomal microarray (array CGH / SNP array) - detects copy number variants genome-wide at high resolution; first-line test for unexplained learning disability, autism with dysmorphism and multiple congenital anomalies. Cannot detect balanced translocations.
• Robertsonian translocation - fusion of two acrocentric chromosomes (e.g. 14;21) - carriers are healthy but have a risk of translocation Down's syndrome in offspring.

Protein Techniques - Immunoassays, Blotting and Flow Cytometry

• ELISA - antibody-based detection of antigens or antibodies (HIV screening, hepatitis serology, autoantibodies, many hormone assays).
• Lateral flow immunoassays - rapid tests (pregnancy, COVID-19 antigen, troponin point-of-care).
• Western blot - proteins separated by size then detected with antibodies (confirmatory tests, e.g. Lyme disease).
• Immunohistochemistry - antibodies on tissue sections (tumour typing - cytokeratins, CD markers, ER/PR/HER2 in breast cancer, PD-L1, MMR proteins).
• Flow cytometry - cells labelled with fluorescent antibodies and counted (CD markers): leukaemia and lymphoma immunophenotyping, CD4 count in HIV, PNH (loss of CD55/CD59), primary immunodeficiency.
• Mass spectrometry - small molecules (drugs, steroids, newborn screening), increasingly proteins and bacterial identification (MALDI-TOF in microbiology).
• Electrophoresis - serum protein electrophoresis (paraproteins), haemoglobin electrophoresis/HPLC (sickle cell, thalassaemia).

mRNA, CRISPR and Gene Therapy

• mRNA vaccines (COVID-19, RSV) - lipid nanoparticle-encapsulated mRNA → host cells make the antigen protein → immune response; mRNA does not enter the nucleus or alter DNA; rare myocarditis (young men, after the second dose).
• Antisense oligonucleotides and siRNA - nusinersen (SMA - modifies SMN2 splicing), inclisiran (siRNA against PCSK9), patisiran (hereditary ATTR amyloidosis).
• Gene therapy - adeno-associated virus vectors deliver functional genes (onasemnogene abeparvovec for SMA; haemophilia gene therapies); lentiviral ex vivo modification of stem cells.
• CRISPR-Cas9 - guide RNA directs Cas9 to cut a specific DNA sequence for editing; exagamglogene autotemcel (Casgevy) - the first CRISPR therapy licensed in the UK (2023) for sickle cell disease and transfusion-dependent β-thalassaemia (reactivates fetal haemoglobin).
• CAR-T cell therapy - patient's T cells engineered with chimeric antigen receptors (e.g. anti-CD19) for B-cell malignancies; cytokine release syndrome and neurotoxicity (ICANS).
• Ethical issues - germline editing (prohibited in the UK), cost and access, consent, long-term safety.

At a Glance

Need	Technique
Detect an RNA virus	RT-PCR
Viral load	Quantitative (real-time) PCR
Single known familial variant	Sanger sequencing
Many genes / undiagnosed rare disease	NGS panel, exome or genome
Microdeletion (22q11.2) or HER2 amplification	FISH
Unexplained learning disability with dysmorphism	Chromosomal microarray
Trisomy	Karyotype (or QF-PCR rapidly)
Leukaemia phenotype, CD4 count, PNH	Flow cytometry
Paraprotein	Serum protein electrophoresis

Spot the Technique

• Rapid confirmation of trisomy 21 in a newborn. → QF-PCR (rapid aneuploidy test), then karyotype for translocation.
• Determining HER2 status in an equivocal breast cancer. → FISH (gene amplification) after IHC 2+.
• Monitoring response to imatinib in CML. → Quantitative RT-PCR for BCR-ABL transcripts.
• A child with developmental delay, normal karyotype. → Chromosomal microarray (then genome sequencing).
• A patient with haemolysis, thrombosis and pancytopenia. → Flow cytometry for PNH (CD55/CD59 loss).

Exam-style Scenarios

• Scenario. A 45-year-old with colorectal cancer has a mother with endometrial cancer at 50. The tumour shows loss of MSH2 protein on immunohistochemistry. What is likely and what follows?
  Answer: Lynch syndrome (mismatch repair deficiency). Germline genetic testing, cascade testing of relatives, colonoscopic surveillance every 2 years from age 25 (MLH1/MSH2), aspirin for chemoprevention, and gynaecological risk-reducing options. MSI-high tumours may respond to immune checkpoint inhibitors.
• Scenario. A COVID-19 PCR remains positive with a Ct of 36 three weeks after recovery. Is the patient infectious?
  Answer: Probably not - a high Ct indicates very little viral RNA, and PCR detects non-viable fragments after recovery. Interpret with symptoms, timing and local infection control guidance.
• Scenario. Why do women with BRCA-mutated ovarian cancer respond to olaparib?
  Answer: Synthetic lethality - BRCA-deficient tumour cells cannot repair double-strand breaks by homologous recombination; PARP inhibition blocks single-strand break repair, leading to lethal accumulation of damage in tumour cells, while normal cells (with one functional BRCA copy) survive.

Key Points

• Know which repair defect causes which syndrome: mismatch - Lynch; NER - xeroderma pigmentosum; HR - BRCA.
• Frameshift (Duchenne) versus in-frame (Becker) deletions explain severity.
• Trinucleotide repeat disorders show anticipation.
• PCR detects nucleic acid, not viability; Ct is inversely related to load.
• Match the test to the question: karyotype, FISH, microarray, panel, exome or genome.
• Imprinting: Prader–Willi (paternal loss), Angelman (maternal loss).

Further Reading

• NHS England: National Genomic Test Directory; Genomic Medicine Service
• Strachan T, Read A. Human Molecular Genetics
• NICE DG27: Molecular testing strategies for Lynch syndrome in people with colorectal cancer
• MHRA (2023): Casgevy (exagamglogene autotemcel) marketing authorisation
• Association for Clinical Genomic Science: Best practice guidelines for variant classification