58-59. Molecular Genetics I-II | Cytogenetics and Gene Amplification

A 28-year-old woman with recurrent miscarriages undergoes genetic testing. Chromosomal analysis reveals a balanced translocation between chromosomes 1 and X. Which technique would most reliably identify this abnormality?

A. G-banding karyotype
B. FISH with locus-specific probes
C. SKY (spectral karyotyping)
D. Array CGH
E. PCR with gene-specific primers

C

• A. G-banding karyotype – Can detect large rearrangements but lacks color distinction; difficult to identify complex translocations.

• B. FISH with locus-specific probes – Detects specific deletions/duplications, but requires prior knowledge of the target locus.

• C. SKY (spectral karyotyping) – Correct. Uses chromosome-painting probes with unique colors, ideal for detecting balanced translocations like between chromosomes 1 and X.

• D. Array CGH – Detects copy number changes (duplications/deletions), but not balanced translocations.

• E. PCR with gene-specific primers – Detects small mutations, not whole-chromosome rearrangements.

A fetus is suspected to have trisomy 21 after abnormal ultrasound findings. The obstetrician needs a rapid, high-resolution test that can be performed directly on amniotic fluid cells without cell culture. Which test is most appropriate?

A. Standard karyotyping with G-banding
B. Metaphase FISH
C. Interphase FISH
D. SKY (spectral karyotyping)
E. Whole genome sequencing

C

• A. Karyotyping – Requires cell culture and metaphase arrest, so not rapid.

• B. Metaphase FISH – Needs dividing cells, not useful for rapid testing.

• C. Interphase FISH – Correct. Works on non-dividing cells, fast, can be done on amniotic fluid, commonly used for trisomy detection.

• D. SKY – Requires metaphase spreads, slower.

• E. Whole genome sequencing – High resolution but time-consuming and expensive.

A 6-year-old boy presents with developmental delay. Array-based comparative genomic hybridization (aCGH) shows a duplication involving the MECP2 gene on Xq28. How does aCGH detect this abnormality?

A. Identifies translocations by painting chromosomes with fluorochromes
B. Measures relative fluorescent probe intensity compared to a control genome
C. Detects single-nucleotide changes using allele-specific primers
D. Identifies trinucleotide repeat expansions by fragment size analysis
E. Detects uniparental disomy using microsatellite markers

B

• A. SKY – Detects translocations, not copy number gains.

• B. aCGH – Correct. Measures patient vs control fluorescent intensity to identify duplications/deletions.

• C. SNP detection – Done by allele-specific PCR, not aCGH.

• D. Trinucleotide repeats – Tested with Southern blot or PCR fragment analysis.

• E. Uniparental disomy – Requires microsatellite or SNP analysis, not aCGH.

A molecular biologist is attempting to clone a human gene into a plasmid vector. The plasmid and the human DNA fragment are cut with EcoRI, producing sticky ends, and then ligated. Which feature ensures that only bacteria containing the recombinant plasmid survive?

A. Presence of an origin of replication in the plasmid
B. Use of ampicillin resistance gene in the plasmid vector
C. Addition of DNA polymerase during ligation
D. Expression of the cloned gene in bacteria
E. Screening for plasmids with blunt ends only

B

• A. Origin of replication – Needed for plasmid replication but does not select bacteria.

• B. Ampicillin resistance – Correct. Ensures that only bacteria with plasmid survive when plated on antibiotic media.

• C. DNA polymerase – Not needed for ligation (DNA ligase is used).

• D. Gene expression – Useful for protein production, but not for survival selection.

• E. Blunt-end cloning – Possible but not selective.

A research scientist wants to produce large quantities of recombinant human insulin in E. coli. Which plasmid feature allows transcription and translation of the inserted human gene into protein?

A. Antibiotic-resistance marker
B. Promoter sequence upstream of the insertion site
C. Origin of replication
D. Restriction enzyme recognition site
E. Operator sequence

B

• A. Antibiotic marker – Enables selection of transformants; doesn’t drive expression. ❌

• B. Promoter sequence – Provides transcriptional control so RNA polymerase initiates mRNA → protein expression. ✅

• C. Ori – Allows plasmid replication, not expression per se. ❌

• D. Restriction site – Enables cloning/insert placement; not sufficient for expression. ❌

• E. Operator – Regulatory element; helpful but needs a promoter; by itself doesn’t ensure expression. ❌

A pathologist analyzes a bone-marrow biopsy and suspects a chromosomal translocation between chromosomes 9 and 22 (BCR-ABL fusion). Which combination of techniques would provide both localization and confirmation of this abnormality?

A. Karyotyping followed by FISH
B. PCR alone
C. aCGH followed by karyotyping
D. SKY alone
E. Whole-genome sequencing alone

A

• A. Karyotyping + FISH – Karyotype shows the balanced translocation; FISH with BCR/ABL probes localizes/confirms the fusion. ✅

• B. PCR alone – May detect fusion transcript but lacks chromosomal localization/visualization. ❌

• C. aCGH + karyotype – aCGH misses balanced events; adding karyotype still benefits from FISH for precise localization. ❌

• D. SKY alone – Can color-paint translocations but typically confirmed with locus-specific FISH for clinical certainty. ❌

• E. WGS alone – Technically possible but impractical/costly for routine confirmation; standard workup is karyotype ± FISH. ❌

A 43-year-old woman with relapsed breast cancer undergoes tumor profiling that reveals hundreds of up-regulated genes related to cell-cycle regulation. The test utilized hybridization of fluorescently labeled complementary DNA samples to thousands of oligonucleotides immobilized on a chip.
Which of the following is a major disadvantage of this method compared with quantitative PCR?

A. Low specificity for mutations
B. High cost and requirement for large sample volumes
C. Inability to detect changes in gene expression
D. Need for endogenous control genes
E. Low throughput

B

• DNA microarray (context): Your slides list its advantages and disadvantages:

  • Advantages — high-throughput, automated, simultaneous analysis.

  • Disadvantages — “Expensive” and “Requires high sample volume; less useful for low-volume testing”.

• (A) Not true — microarrays have high specificity for polymorphisms.

• (C) Incorrect — detecting expression changes is the purpose of this method.

• (D) Incorrect — qPCR requires control genes; microarrays do not.

• (E) Incorrect — microarrays have very high throughput.

A 7-year-old boy presents with failure to thrive and recurrent infections. Bone-marrow-derived stem cells are harvested, infected ex vivo with a viral vector carrying a normal gene copy, and reinfused. During follow-up, he develops leukemia due to insertion of the therapeutic gene near an oncogene.
Which of the following vectors was most likely used?

A. Adeno-associated virus (AAV)
B. Naked DNA
C. Retrovirus
D. Liposome
E. CRISPR-Cas9 complex

C

• Retroviral vectors (C): Integrate into the host genome, creating risk of insertional mutagenesis (leukemia cases in early ADA-SCID trials).

• AAV (A): Generally non-integrating, lower mutagenesis risk.

• Naked DNA (B): Rarely efficient for stable expression.

• Liposome (D): Used for transient delivery; low genomic integration risk.

• CRISPR-Cas9 (E): Editing tool; not a vector.