Molecular Diagnostics
What to master
Follow the workflow: specimen, nucleic acid extraction, amplification or hybridization, detection, interpretation. Know polymerase chain reaction stages and real-time chemistry (hydrolysis probes, molecular beacons, intercalating dyes), and that the cycle threshold is inversely proportional to starting target; roughly one cycle equals a two-fold difference. Reverse transcription PCR for RNA targets, digital PCR for absolute quantitation, Sanger sequencing for single variants, next-generation sequencing for panels, FISH and karyotype for structural and copy-number changes, and microarray for genome-wide copy number. Distinguish Southern (DNA), northern (RNA), and western (protein) blotting.
Quality and contamination control: positive and negative controls, no-template control, internal extraction and inhibition controls, unidirectional workflow, aliquoted reagents, and uracil-N-glycosylase systems. Common inhibitors include heme, heparin, melanin, and urea. Validation covers limit of detection, precision, reportable range, and reference range. Applications include viral load monitoring, oncology mutation testing, inherited disease, HLA typing, pharmacogenomics, cell-free DNA screening, and blood group genotyping.
Traps
Distinguish true positive from amplicon contamination, and remember that a variant under a primer or probe binding site can cause allelic dropout and a false-negative result.
Worked example
Sample A has a cycle threshold of 22 and sample B has 26. The difference is 4 cycles, so sample A contains approximately 2^4, or 16 times, more target nucleic acid than sample B.