CSMLS Molecular Diagnostics 1 — Questions and Answers
Question 1: What is the primary purpose of the denaturation step in PCR?
- To separate double-stranded DNA into single strands (Correct answer)
- To allow primers to bind to the template
- To synthesize new DNA strands using polymerase
- To remove RNA contamination from the sample
Correct answer: To separate double-stranded DNA into single strands
Denaturation at 94–96°C breaks the hydrogen bonds between complementary base pairs, separating the double-stranded DNA into single-stranded templates for subsequent primer annealing.
Question 2: Why is Taq polymerase used in PCR instead of a standard DNA polymerase?
- It has higher fidelity and fewer errors
- It is thermostable and survives repeated heating to 94°C (Correct answer)
- It can amplify RNA directly without reverse transcription
- It requires no cofactors or dNTPs
Correct answer: It is thermostable and survives repeated heating to 94°C
Taq polymerase, derived from Thermus aquaticus, is thermostable and retains activity through the repeated high-temperature denaturation cycles required in PCR.
Question 3: In quantitative real-time PCR (qPCR), what does the Ct (cycle threshold) value represent?
- The total number of PCR cycles programmed
- The cycle at which fluorescence crosses the detection threshold (Correct answer)
- The melting temperature of the amplified product
- The concentration of primers in the reaction
Correct answer: The cycle at which fluorescence crosses the detection threshold
The Ct value is the PCR cycle number at which the fluorescence signal rises above background threshold; a lower Ct indicates a higher initial amount of target nucleic acid.
Question 4: Which technique is used to detect RNA viruses such as HIV or Hepatitis C virus by molecular methods?
- Southern blotting
- Reverse transcriptase PCR (RT-PCR) (Correct answer)
- Pulsed-field gel electrophoresis
- Loop-mediated isothermal amplification
Correct answer: Reverse transcriptase PCR (RT-PCR)
RT-PCR uses reverse transcriptase to first convert the viral RNA into complementary DNA (cDNA), which is then amplified by standard PCR to enable detection of RNA-based targets.
Question 5: SYBR Green dye in qPCR generates signal by:
- Binding specifically to the primer-template junction
- Intercalating into any double-stranded DNA formed during amplification (Correct answer)
- Hybridizing to a specific probe sequence on the amplicon
- Fluorescing when cleaved by the 5'-to-3' exonuclease activity of Taq
Correct answer: Intercalating into any double-stranded DNA formed during amplification
SYBR Green intercalates non-specifically into any double-stranded DNA, producing fluorescence proportional to the amount of double-stranded PCR product accumulated.
Question 6: Fluorescence in situ hybridization (FISH) is primarily used in molecular diagnostics to:
- Amplify specific gene sequences for sequencing
- Detect specific DNA sequences or chromosomal rearrangements in intact cells (Correct answer)
- Quantify mRNA expression levels in tissue samples
- Determine antibiotic resistance profiles of bacteria
Correct answer: Detect specific DNA sequences or chromosomal rearrangements in intact cells
FISH uses fluorescently labeled DNA probes that hybridize to complementary chromosomal sequences, enabling visualization of gene deletions, amplifications, or translocations directly in cells or tissue sections.
Question 7: Which electrophoresis technique is best suited for separating very large DNA fragments greater than 50 kilobases?
- Agarose gel electrophoresis
- Polyacrylamide gel electrophoresis
- Pulsed-field gel electrophoresis (PFGE) (Correct answer)
- Capillary electrophoresis
Correct answer: Pulsed-field gel electrophoresis (PFGE)
PFGE uses alternating electrical fields that cause large DNA molecules to reorient and migrate differently, enabling resolution of fragments up to several megabases that co-migrate in standard agarose electrophoresis.
CSMLS Molecular Diagnostics 1
What is the primary purpose of the denaturation step in PCR?