Lab Techniques & Genetics Concepts — USMLE Step 1 Notes

Amplification & editing

• PCR (polymerase chain reaction): amplifies a DNA target through cycles of denaturation, annealing of primers, and extension by a heat-stable polymerase; used for diagnosis, detecting infections/mutations and forensics. Reverse-transcription PCR (RT-PCR) starts from RNA; quantitative PCR measures copy number (e.g. viral load).
• CRISPR/Cas9: a guide RNA directs the Cas9 nuclease to a matching DNA sequence to cut it, allowing targeted gene editing (knockout or, with a template, correction). Adapted from a bacterial adaptive-immune system; now an approved therapy for sickle cell disease/beta-thalassaemia.

Blotting techniques

• SNoW DRoP: Southern = DNA, Northern = RNA, Western = protein (with labelled antibodies).
• Southwestern blot: detects DNA-binding proteins (e.g. transcription factors) using labelled DNA probes.

Cell & protein assays

• Flow cytometry: lasers analyse single cells tagged with fluorescent antibodies to count and characterise them by surface markers (e.g. CD4 counts in HIV, immunophenotyping leukaemias, PNH testing for CD55/CD59).
• ELISA: uses antibody-antigen binding with an enzyme-linked colour readout to detect and quantify a specific protein/antibody (e.g. HIV screening, hormone assays).
• Karyotyping: visualises whole chromosomes (number and gross structure) from dividing cells to detect aneuploidy and large translocations/deletions; FISH detects specific sequences with fluorescent probes.

Gene-silencing RNAs

• microRNA (miRNA): small endogenous RNAs that base-pair (often imperfectly) with target mRNAs to repress translation or promote degradation, fine-tuning gene expression; dysregulation contributes to cancer.
• siRNA (small interfering RNA): typically exogenous double-stranded RNA that guides near-perfect cleavage of a matching mRNA (RNA interference); the basis of a growing class of therapeutics.

Population & inheritance concepts

• Hardy-Weinberg: for two alleles, p + q = 1 and p² + 2pq + q² = 1; disease-allele frequency lets you estimate carrier (2pq) and affected (q²) proportions, assuming no selection, migration, mutation, mating bias or drift.
• Allelic vs locus heterogeneity: allelic = different mutations in the SAME gene cause the phenotype (e.g. beta-thalassaemia); locus = mutations in DIFFERENT genes cause the same phenotype (e.g. retinitis pigmentosa).
• Mosaicism: two genetically different cell lines from a post-zygotic mutation (somatic mosaicism, e.g. McCune-Albright; gonadal mosaicism can transmit disease from unaffected parents).
• Heteroplasmy: a mix of normal and mutant mitochondria in a cell — explains the variable severity of mitochondrial disease.
• Uniparental disomy: both copies of a chromosome come from ONE parent; with imprinted regions it can cause Prader-Willi or Angelman even without a deletion.

Imprinting: Prader-Willi vs Angelman

• Both involve chromosome 15q. Prader-Willi = loss of the paternal genes (paternal deletion or maternal uniparental disomy): hyperphagia/obesity, hypotonia, hypogonadism, intellectual disability.
• Angelman = loss of the maternal gene (UBE3A; maternal deletion or paternal uniparental disomy): ataxic 'happy puppet' gait, inappropriate laughter, seizures, severe intellectual disability.

Mitochondrial disorders

• Maternally inherited with heteroplasmy. Mitochondrial myopathies show 'ragged red fibres' on muscle biopsy with exercise intolerance and lactic acidosis; MELAS (encephalopathy, lactic acidosis, stroke-like episodes) and MERRF (myoclonic epilepsy) are classic.
• Leber hereditary optic neuropathy (LHON): subacute bilateral central vision loss in young men from mitochondrial DNA mutations affecting complex I.

Named syndromes

• McCune-Albright syndrome: post-zygotic activating GNAS mutation (mosaic) → the triad of polyostotic fibrous dysplasia, cafe-au-lait macules with irregular ('coast of Maine') borders, and precocious puberty/other endocrinopathies; not inherited (lethal if germline).
• Williams syndrome: microdeletion of chromosome 7 (including elastin) → 'elfin' facies, an extremely friendly/social personality, intellectual disability, supravalvular aortic stenosis and hypercalcaemia.
• Robertsonian translocation: fusion of two acrocentric chromosomes (13, 14, 15, 21, 22); a balanced carrier is healthy but at risk of offspring with translocation Down syndrome (21) or Patau (13).
• Acrodermatitis enteropathica: autosomal recessive zinc malabsorption (SLC39A4) → periorificial and acral dermatitis, alopecia and diarrhoea; treat with zinc.

Genetic disorder by chromosome (quick map)

• 3 von Hippel-Lindau; 4 Huntington, achondroplasia (FGFR3); 5 cri-du-chat (5p deletion), familial adenomatous polyposis (APC); 6 haemochromatosis (HFE); 7 cystic fibrosis (CFTR), Williams; 9 Friedreich ataxia (FXN), tuberous sclerosis (TSC1); 11 beta-globin (sickle cell/thalassaemia), Wilms/WT1, MEN1.
• 13 Patau, Wilson (ATP7B), BRCA2, retinoblastoma (RB1); 15 Prader-Willi/Angelman, Marfan (FBN1), Tay-Sachs; 16 ADPKD (PKD1), alpha-globin; 17 neurofibromatosis type 1, BRCA1, TP53; 18 Edwards; 21 Down, amyloid precursor protein; 22 neurofibromatosis type 2, DiGeorge (22q11); X fragile X, Duchenne, haemophilia, G6PD.

High-yield revision notes; verify against a primary source before clinical use.