Molecular diagnostics Practice Questions

Correct answer: Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR) uses thermal cycling to denature, anneal primers, and extend DNA in cycles, resulting in exponential amplification of the target DNA segment — a foundational molecular diagnostic tool.

Correct answer: Real-Time (quantitative) PCR (qPCR)

Real-Time (quantitative) PCR (qPCR) uses fluorescent probes or dyes to measure DNA amplification as it happens, enabling quantification of DNA or RNA starting amounts in a sample.

Correct answer: Array comparative genomic hybridization (aCGH)

Array comparative genomic hybridization (aCGH) compares patient DNA to reference DNA at many loci across the genome and detects copy number changes (deletions or duplications) with much higher resolution than conventional karyotyping.

Correct answer: Sanger DNA sequencing

Sanger DNA sequencing reads the exact nucleotide order of DNA and can detect single-nucleotide variants or small INDELs in a gene, making it the gold-standard for targeted mutation detection.

Correct answer: DNA / gene expression microarray

Multiplex PCR uses multiple primer sets in a single reaction tube to amplify several different DNA sequences at once, saving time and reagents when screening for multiple targets.

Correct answer: Western blotting with specific antibodies

Western blotting separates proteins by size via electrophoresis, transfers them to a membrane, and uses antibodies to detect specific proteins in a complex mixture — making it a standard protein-level diagnostic method.

Correct answer: Molecular tests examine DNA sequences; cytogenetics examines chromosome structure

Molecular genetic tests focus on specific genes or DNA sequences (point mutations, small deletions or insertions), whereas cytogenetic tests examine large-scale chromosomal structure or number to detect gross chromosomal abnormalities.

Correct answer: PCR detects pathogen DNA even when organisms are non-viable

PCR amplifies genetic material directly from samples, enabling detection of pathogens even if they’re non-viable or present in low numbers, which is especially useful for fastidious or slow-growing organisms.

Correct answer: Gene-expression (DNA) microarray

Gene expression microarrays measure the relative expression levels of thousands of genes simultaneously, allowing for profiling of global transcriptional changes in tumor samples.

Correct answer: It needs gel electrophoresis to detect products

Conventional PCR requires post-PCR processing (gel electrophoresis) to visualize amplification products, which is time-consuming and increases contamination risk — a limitation addressed by real-time PCR with in-cycle fluorescence detection.

Correct answer: TaqMan qPCR assay

TaqMan qPCR uses a dual-labeled hydrolysis probe: during PCR, the Taq polymerase cleaves the probe if hybridized to target DNA, releasing fluorescence and providing highly specific quantitative detection.

Correct answer: Next-generation (high-throughput) sequencing

Next-generation sequencing (NGS) can sequence large numbers of DNA fragments in parallel and detect mutations (including indels) even if present at low allele frequency within a heterogeneous cell population, unlike Sanger sequencing which has lower sensitivity in mixed samples.

Correct answer: Fluorescence in situ hybridization (FISH)

FISH uses fluorescent DNA probes that bind to specific chromosomal regions; it can visualize rearrangements such as translocations directly in cells, making it ideal for detecting chromosomal translocations in leukemia.

Correct answer: It work at a constant temperature with simple equipment

Isothermal amplification methods such as LAMP or RPA amplify nucleic acids at a constant temperature, eliminating the need for a thermal cycler; this makes them well suited for rapid, low-cost, point-of-care diagnostics especially in resource-limited settings.

Correct answer: Reverse-transcription PCR (RT-PCR)

Reverse-transcription PCR (RT-PCR) first converts viral RNA into complementary DNA and then amplifies it, making it the standard method for detecting RNA viruses with high sensitivity and specificity.

Correct answer: Detecting very low amounts of target nucleic acid or pathogen

High analytical sensitivity refers to the capacity of a molecular assay to detect very small amounts of target nucleic acid (or small number of pathogen copies), enabling early or low-level detection of infections or mutations.

Correct answer: DNA microarray expression profiling

DNA microarray-based expression profiling can measure mRNA levels of thousands of genes simultaneously and, depending on the array design, may also detect variant sequences — giving a broad overview of gene expression and potential mutations.

Correct answer: To find mutations or rearrangements guiding targeted therapy

Molecular diagnostics in oncology aims to detect specific genetic mutations, copy-number changes, or gene rearrangements in tumors — information that can guide targeted therapies, prognosis, and personalized treatment plans.

Correct answer: Need for specialized equipment and higher cost

Molecular diagnostic methods often require specialized equipment (e.g., thermal cyclers, sequencers), trained personnel, and can be more expensive to set up than traditional assays, which can limit their availability in some settings.

Correct answer: MLPA or a copy-number microarray assay

Techniques like MLPA or copy-number arrays (e.g., aCGH) are designed to detect deletions or duplications of exons or gene regions (copy-number variations), which sequencing may miss due to limitations in quantifying gene dosage.

Correct answer: Multiplex PCR or multiplex qPCR

Multiplex PCR (or multiplex qPCR) uses multiple primer pairs or probes in one reaction to simultaneously amplify multiple target sequences — ideal for rapid detection of several pathogens or resistance genes in a single sample.