ASCP Molecular Diagnostics 2 — Questions and Answers

Question 1: In PCR, what is the role of the thermostable DNA polymerase (Taq polymerase)?

To denature double-stranded DNA
To synthesize new DNA strands using primers as starting points (Correct answer)
To ligate DNA fragments together
To digest RNA in the reaction

Correct answer: To synthesize new DNA strands using primers as starting points

Taq polymerase (from Thermus aquaticus) is a thermostable DNA polymerase that synthesizes new DNA strands by extending from the 3' end of primers using the template strand. Its heat stability allows repeated thermocycling.

PCR requires three temperature steps: denaturation at approximately 95 degrees C, annealing at approximately 50–65 degrees C, and extension at approximately 72 degrees C. Taq polymerase survives the 95 degree denaturation step, allowing reuse over 30–40 cycles. Taq lacks 3' to 5' proofreading exonuclease activity with a high error rate; high-fidelity polymerases like Pfu and Q5 have proofreading capability. Taq adds a single adenine overhang, enabling TA cloning. Hot-start Taq with antibody blocking prevents non-specific amplification during setup.

Question 2: Southern blotting detects:

RNA sequences
Protein expression
DNA sequences (Correct answer)
Lipid biomarkers

Correct answer: DNA sequences

Southern blotting (developed by Edwin Southern) detects specific DNA sequences. DNA is digested with restriction enzymes, separated by gel electrophoresis, transferred to a membrane, and hybridized with a labeled probe.

Southern blotting steps: restriction enzyme digestion, gel electrophoresis, denaturation, transfer to nylon or nitrocellulose membrane, hybridization with labeled probe (radioactive or chemiluminescent), and autoradiography or imaging. Applications include fragile X syndrome (triplet repeat expansion), BCR-ABL rearrangement, gene deletion or duplication analysis, and restriction fragment length polymorphism (RFLP) analysis. Southern blotting has been largely replaced by PCR and NGS for most applications but remains used for characterizing large structural variants.

Question 3: A FISH break-apart probe is designed to:

Detect gene amplification by counting signal copies
Detect gene rearrangements by showing split signals when a gene is disrupted (Correct answer)
Measure mRNA expression level
Identify chromosomal aneuploidy only

Correct answer: Detect gene rearrangements by showing split signals when a gene is disrupted

Break-apart FISH probes flank a gene of interest with differently colored labels. In the normal state, signals are adjacent or fused. A chromosomal rearrangement splits the signals apart, indicating rearrangement regardless of the partner gene.

Break-apart probes are useful when a gene rearranges with multiple possible partners, such as ALK in lung cancer, MYC in lymphoma, and SS18 in synovial sarcoma. The 5' probe (one color) and 3' probe (another color) flank the breakpoint. Normal: two fused or closely adjacent signals. Rearranged: one split signal showing one 5' color separated from the 3' color. Dual-fusion probes are used when specific translocation partners are known, such as BCR-ABL. Amplification probes count signals where more than two per cell indicates copy number gain.

Question 4: Pharmacogenomic testing for CYP2D6 variants is clinically important because CYP2D6 metabolizes:

Warfarin
A large number of drugs including codeine, tamoxifen, and many antidepressants (Correct answer)
Metformin
Penicillin antibiotics

Correct answer: A large number of drugs including codeine, tamoxifen, and many antidepressants

CYP2D6 metabolizes approximately 25% of clinically used drugs, including codeine (to morphine), tamoxifen (to endoxifen), tricyclic antidepressants, SSRIs, and antipsychotics. Genetic variants create poor, intermediate, normal, rapid, and ultrarapid metabolizer phenotypes.

CYP2D6 phenotypes: Poor metabolizers (PMs, approximately 7% of Caucasians) have two non-functional alleles (*4, *5); drugs accumulate causing toxicity. Ultrarapid metabolizers (UMs, approximately 2% of Caucasians, over 20% in North Africa) have gene duplication; drugs clear too fast causing inadequate effect. Clinical implications include codeine in UMs causing excessive morphine and respiratory depression risk (FDA black box warning), and tamoxifen in PMs resulting in reduced endoxifen and worse breast cancer outcomes.

Question 5: Microsatellite instability (MSI) testing is most relevant for which condition?

Factor V Leiden thrombophilia
Lynch syndrome (hereditary nonpolyposis colorectal cancer) (Correct answer)
BRCA1 hereditary breast cancer
ABO blood group determination

Correct answer: Lynch syndrome (hereditary nonpolyposis colorectal cancer)

Lynch syndrome is caused by germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2). Deficient MMR causes MSI — length changes in repetitive DNA sequences. MSI-H tumors in young patients prompt Lynch syndrome evaluation.

Microsatellites are 1–6 bp repeating sequences prone to polymerase slippage errors. MMR proteins (MLH1-PMS2 heterodimer and MSH2-MSH6 heterodimer) correct these errors. Loss of MMR leads to MSI and accumulating frameshift mutations genome-wide. The Bethesda panel uses markers BAT25, BAT26, D5S346, D2S123, and D17S250. MSI-H means two or more of five markers are unstable. MSI-H is predictive of immunotherapy (pembrolizumab) response and is FDA-approved pan-tumor. It is used for Lynch syndrome screening in colorectal and endometrial cancer.

Question 6: In the CRISPR-Cas9 system, what is the function of the guide RNA (gRNA)?

To code for the Cas9 protein
To direct the Cas9 nuclease to a specific DNA sequence via complementary base pairing (Correct answer)
To repair the double-strand break
To transcribe the target gene

Correct answer: To direct the Cas9 nuclease to a specific DNA sequence via complementary base pairing

The gRNA contains a approximately 20 nucleotide spacer sequence complementary to the target DNA. It directs Cas9 to the correct genomic location adjacent to a PAM (protospacer adjacent motif, 5'-NGG-3') sequence, where Cas9 creates a double-strand break.

The CRISPR-Cas9 system adapted from Streptococcus pyogenes uses a single guide RNA (fusion of crRNA and tracrRNA). The 20 nucleotide spacer sequence base-pairs with the target DNA strand; Cas9 requires an NGG PAM on the non-target strand immediately 3' of the protospacer. Cas9 creates a double-strand break via the HNH and RuvC nuclease domains. Cellular repair proceeds through NHEJ (error-prone, causing indels and gene knockout) or HDR (template-mediated precise editing). Diagnostic applications include SHERLOCK and DETECTR systems using Cas12a or Cas13 for rapid pathogen detection.

ASCP Molecular Diagnostics 2

In PCR, what is the role of the thermostable DNA polymerase (Taq polymerase)?