Molecular Techniques and Methods
Core concepts
Know the principle, strengths, limitations, and failure modes of each platform. PCR and real-time PCR (threshold cycle, melt curve, efficiency), Sanger sequencing (chain termination, dideoxynucleotides, ~700-1000 bp reads), next-generation sequencing (clonal amplification, massively parallel short reads, depth and coverage), and long-read platforms. Understand hybridization methods (Southern, Northern, FISH, microarray), nucleic acid extraction and quality metrics (A260/A280), and the role of controls in each assay.
Be able to explain allele-specific methods (ARMS, TaqMan genotyping), fragment analysis for repeat expansions, MLPA for copy number, bisulfite sequencing for methylation, and digital PCR for absolute quantitation. Know when each is appropriate: Sanger for single variants, NGS for panels and unknown variants, MLPA or array for deletions/duplications, fragment analysis for triplet repeats.
Traps
Questions probe limits of detection, the difference between analytical sensitivity and specificity, and contamination control (uracil-N-glycosylase, unidirectional workflow, negative controls). A frequent trap is confusing a method's ability to detect a variant with its ability to quantify it. Remember that NGS cannot reliably detect large structural variants or repeat expansions without specialized analysis, and that low-level mosaicism requires adequate depth.
Rule of thumb
Match the method to the variant class: substitution -> sequencing or allele-specific assay; small insertion/deletion -> sequencing; copy number -> MLPA/array/digital PCR; repeat expansion -> fragment analysis or Southern blot; methylation -> bisulfite methods.
Worked example
A 5% variant allele fraction in a tumor requires a method with limit of detection below 5%; Sanger (~15-20%) is inadequate, whereas deep NGS or digital PCR is appropriate.