Recombinant DNA Technology (PCR, CRISPR, Microarray, Genomics) MCQs

Biochemistry · 39 free questions with answers & explanations.

1. A research laboratory uses Southern blotting to detect a specific gene deletion. Which sequence of steps correctly describes the Southern blot procedure?
2. CRISPR-Cas9 genome editing requires two essential components. The Cas9 endonuclease is guided to the target DNA sequence by:
3. Real-time quantitative PCR (qRT-PCR) is used to quantify mRNA expression levels. In this technique, cDNA is first synthesized from mRNA using reverse transcriptase. The SYBR Green method detects PCR product by binding to double-stranded DNA. A fundamental limitation of SYBR Green compared to TaqMan probe-based detection is:
4. CRISPR-Cas9 genome editing uses guide RNA (gRNA) to direct the Cas9 nuclease to a specific genomic locus. After creating a double-strand break (DSB), the cell repairs the break via two major pathways. For therapeutic gene correction in sickle cell disease, which repair pathway would be deliberately utilized to achieve precise correction of the HBB beta-6 codon?
5. In CRISPR-Cas9 gene editing, the guide RNA (sgRNA) directs Cas9 to the target DNA. Cas9 requires a protospacer adjacent motif (PAM) sequence (5'-NGG-3' for SpCas9) immediately 3' of the target for cleavage. What is the biochemical rationale for this PAM requirement?
6. Quantitative PCR (qPCR/RT-qPCR) uses a fluorescent probe strategy. In TaqMan assays, a dual-labeled probe is used. What is the mechanism by which fluorescence increases only during legitimate amplification?
7. Fluorescence in situ hybridization (FISH) detects specific DNA sequences in chromosomal spreads or interphase nuclei. In a patient with suspected BCR-ABL1 fusion (CML), dual-fusion FISH (D-FISH) is preferred over break-apart FISH. Why?
8. In real-time quantitative PCR (qPCR) using TaqMan probes, the probe is labeled with a fluorophore at the 5' end and a quencher at the 3' end. During extension, Taq polymerase's 5'-to-3' exonuclease activity degrades the probe, separating fluorophore from quencher. Fluorescence increases proportional to PCR product amount. A laboratory receives a sample where the qPCR amplification curve shows a high cycle threshold (Ct value of 38 out of 40 cycles). What does this indicate?
9. In DNA microarray (gene chip) technology, thousands of oligonucleotide probes are spotted on a solid surface. Patient mRNA is reverse-transcribed to cDNA, labeled with fluorescent dyes, and hybridized to the array. In a two-color comparative genomic hybridization (aCGH) for copy number variation detection, tumor DNA is labeled red and normal control DNA is labeled green. A region showing red:green ratio of 2.0 indicates what?
10. Next-generation sequencing (NGS) of a familial breast cancer gene panel shows a variant in BRCA1: c.5266dupC (also designated 5382insC), a frameshift mutation creating a premature stop codon. This variant is common among Ashkenazi Jewish populations. The resulting truncated BRCA1 protein lacks the BRCT domain. The BRCT domain is essential for which specific DNA repair function?
11. In CRISPR-Cas9 gene editing, which component provides the specificity for targeting a particular genomic sequence, and what short DNA motif adjacent to the target is required for Cas9 to cut?
12. In quantitative real-time PCR (qRT-PCR) for gene expression analysis, what is the meaning of the Ct (cycle threshold) value, and what comparison method normalizes gene expression across samples?
13. Southern blotting is used to analyze DNA. A clinical application is confirming Huntington's disease (HD). In HD, a patient's Southern blot shows an expanded band. This expansion represents:
14. Next-generation sequencing (NGS) platforms using sequencing-by-synthesis (SBS) technology generate millions of short reads that require bioinformatic assembly. Which step PRECEDES library amplification and is unique to NGS compared to Sanger sequencing?
15. In quantitative real-time PCR (qRT-PCR) using SYBR Green chemistry, a researcher notices non-specific amplification detected by the presence of multiple peaks on melt curve analysis. What is the most appropriate corrective measure?
16. CRISPR-Cas9 gene editing requires two components: the Cas9 endonuclease and a guide RNA (gRNA). After Cas9 creates a double-strand break (DSB), the cell repairs it via two competing pathways. Which pathway is preferred for precise gene correction, and what is required for this pathway to operate?
17. Whole exome sequencing (WES) captures all protein-coding regions (~1% of the genome). A clinical WES report identifies a variant of uncertain significance (VUS) in the BRCA2 gene. Which additional evidence is MOST useful for reclassifying this VUS as pathogenic?
18. A laboratory wishes to detect a point mutation in BRCA1 that changes a single nucleotide. They design an allele-specific PCR where one primer perfectly matches the mutant allele but has a 3'-mismatch with the wild-type allele. Which molecular property of DNA polymerase makes this test specific for the mutant sequence?
19. CRISPR-Cas9 gene editing creates a double-strand break (DSB) at a target sequence guided by a single-guide RNA (sgRNA). The break is repaired by either NHEJ or HDR. In therapeutic gene correction (e.g., for sickle cell disease), which repair pathway is preferred, and why?
20. Fluorescence in situ hybridisation (FISH) is used to detect chromosomal abnormalities. A patient with suspected Philadelphia chromosome t(9;22) translocation is tested by dual-colour FISH. A positive result shows fusion of BCR (chromosome 22, red) and ABL1 (chromosome 9, green) probes. What would be observed in a positive FISH result?
21. Southern blotting is used to analyse DNA. A restriction enzyme digest of genomic DNA is electrophoresed, transferred to a nylon membrane, and hybridised with a labelled probe. In which clinical scenario is Southern blotting the gold standard diagnostic tool?
22. In CRISPR-Cas9 gene editing, the guide RNA (gRNA) directs Cas9 to cut double-stranded DNA at a specific site. The absolute prerequisite sequence element present in the target DNA (not in the gRNA) is:
23. In real-time PCR (qPCR) using TaqMan probes, fluorescence signal increases are due to which molecular mechanism?
24. Sanger dideoxy sequencing uses chain-terminating dideoxynucleotides (ddNTPs). Why do ddNTPs terminate DNA chain elongation?
25. In next-generation sequencing (NGS), the 'sequencing by synthesis' approach used by Illumina platforms employs reversible terminator chemistry. What differentiates this from Sanger sequencing in terms of scale?
26. CRISPR-Cas9 genome editing uses a guide RNA (sgRNA) to direct Cas9 nuclease to a specific genomic locus. A 3-nucleotide sequence immediately 3' to the target DNA on the non-template strand, required for Cas9 cleavage, is called the:
27. Next-generation sequencing (NGS) platforms achieve massively parallel sequencing. The Illumina platform uses a sequencing-by-synthesis approach that employs which terminator chemistry to achieve single-base resolution with reversible stopping?
28. In CRISPR-Cas9 gene editing, Cas9 creates a double-strand break (DSB) in DNA at a site determined by the guide RNA (gRNA). The two primary pathways for DSB repair differ in their outcomes. Which pathway is preferred for precise gene correction and requires a template?
29. In CRISPR-Cas9 gene editing, the guide RNA (gRNA) must match a target DNA sequence adjacent to which motif for Cas9 cleavage to occur?
30. CRISPR-Cas9 gene editing uses guide RNA to direct Cas9 nuclease. The PAM (protospacer adjacent motif) sequence required for Cas9 cleavage from Streptococcus pyogenes is:
31. Southern blotting is used to detect specific DNA sequences. The correct sequence of steps is:
32. In CRISPR-Cas9 gene editing, guide RNA (gRNA) directs Cas9 nuclease to a target DNA sequence. Which additional DNA sequence is an absolute requirement adjacent to the target for Cas9 to cleave?
33. In allele-specific PCR (AS-PCR) for detecting point mutations (e.g., sickle cell disease HbS mutation), which design principle ensures that the primer amplifies only the mutant but not the wild-type allele?
34. CRISPR-Cas9 gene editing uses guide RNA (gRNA) to direct Cas9 nuclease to specific genomic loci. The molecular requirement that limits Cas9 cutting to the correct target site (preventing off-target editing) includes:
35. CRISPR-Cas9 base editing differs from standard CRISPR-Cas9 nuclease in that base editors:
36. CRISPR-Cas9 genome editing requires a guide RNA (gRNA) to direct Cas9 to the target sequence. The ESSENTIAL genomic sequence feature that determines permissible Cas9 cut sites adjacent to any target is:
37. CRISPR-Cas9 gene editing requires a guide RNA (gRNA) and Cas9 endonuclease. The gRNA directs Cas9 to the target by Watson-Crick base pairing with the DNA strand adjacent to which essential sequence that Cas9 recognises?
38. CRISPR-Cas9 achieves site-specific DNA cleavage by:
39. Southern blotting detects specific DNA sequences. The correct order of steps is: