ASCP MB Exam Prep: 32 Practice Questions + Study Plan
This free MB(ASCP) prep guide follows ASCP BOC's current Scientist in Molecular Biology content guideline (revised December 5, 2025): 100 questions in 2½ hours, weighted toward techniques and clinical applications.
Molecular Science 20–25% · Molecular Techniques 30–35% · Laboratory Operations 15–20% · Applications of Molecular Testing 30–35%
Start with Question 1. Keep a tally of misses by section; your results tell you where to start the study plan below. These are original Castleport questions, not official ASCP items.
32-question MB practice set
Molecular Science — Questions 1–7
1. (MB-SCI-01)
Two 200-bp PCR products are run on a melt curve. Product A is 65% G+C; product B is 35% G+C. Which statement is correct?
Show answer for question 1
Answer: B. G–C pairs form three hydrogen bonds and A–T pairs form two, and G–C-rich sequences also stack more stably. More energy is needed to separate the strands, so the melting temperature (Tm) is higher. A reverses the bond counts. C is wrong because base composition matters as well as length. D reverses the effect. The same idea drives primer Tm and melt-curve genotyping.
Topic: Nucleic acid chemistry (content outline I.A).
Evidence for this item
- MIQE 2.0 qPCR guidelines (2025) — Duplex melting behavior depends on sequence composition and assay conditions; GC content is one determinant.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.A
2. (MB-SCI-02)
A lab needs to measure HIV-1 RNA in plasma by PCR. Which enzyme step is required before PCR amplification can begin?
Show answer for question 2
Answer: B. PCR polymerases copy DNA, so an RNA target must first be copied into complementary DNA (cDNA) by reverse transcriptase, an RNA-dependent DNA polymerase. A: ligase seals DNA ends; it doesn't convert RNA. C: restriction enzymes cut double-stranded DNA at recognition sites. D: destroying the RNA destroys the target.
Topic: Reverse transcriptase (content outline I.C.3).
Evidence for this item
- NCBI Probe: Real-Time qRT-PCR — Reverse transcription converts RNA to cDNA before PCR amplification.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.C.3
3. (MB-SCI-03)
Sequencing shows a single-base substitution that changes the codon CAG (glutamine) to TAG. How is this variant classified by its effect on the protein?
Show answer for question 3
Answer: C. TAG is a stop codon, so the change creates a premature stop: a nonsense variant. A: a silent change keeps the same amino acid. B: a missense change swaps one amino acid for another. D: frameshifts come from insertions or deletions that aren't multiples of three, not from a single-base substitution.
Topic: Mutation type (content outline I.A.5).
Evidence for this item
- NCBI Bookshelf: mutation classes — A nonsense mutation creates a premature termination codon.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.A.5
4. (MB-SCI-04)
In one family, an affected mother has passed a disorder to all of her children. An affected man in the same family has no affected children. Which inheritance pattern best fits?
Show answer for question 4
Answer: C. Mitochondrial DNA comes from the mother through the egg's cytoplasm. An affected mother can pass it to all of her children; an affected father passes it to none. Severity can vary because of heteroplasmy (a mix of normal and variant mtDNA). A would let affected fathers pass it on. B would not fit an affected mother passing it to every child of both sexes. D would not produce transmission from one affected parent to every child.
Topic: Mitochondrial inheritance (content outline I.B.6).
Evidence for this item
- GeneReviews: Primary Mitochondrial Disorders Overview — Mitochondrial DNA is maternally inherited.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.B.6
5. (MB-SCI-05)
A variant changes the invariant GT dinucleotide at the 5′ end of an intron. What is the most likely consequence?
Show answer for question 5
Answer: B. The 5′ (donor) splice site starts with GT in the intron. Breaking it often makes the spliceosome skip the nearby exon or use a cryptic splice site. A: intron sequence isn't translated. C: the polyadenylation signal sits near the 3′ end of the transcript. D: splice-site changes don't raise transcription.
Topic: Splicing (content outline I.B.3).
Evidence for this item
- IMGT Education: Splicing sites — The canonical 5′ intron donor begins GT in DNA (GU in pre-mRNA); disruption can impair splicing.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.B.3
6. (MB-SCI-06)
During DNA replication, which enzyme seals the nicks between Okazaki fragments on the lagging strand?
Show answer for question 6
Answer: C. DNA polymerase can only add nucleotides in the 5′→3′ direction, so the lagging strand is made in short Okazaki fragments. After the RNA primers are replaced with DNA, DNA ligase forms the final phosphodiester bond that joins them. A: helicase unwinds the helix. B: primase lays down RNA primers. D: reverse transcriptase copies RNA into DNA and isn't part of normal chromosomal replication.
Topic: Replication and ligase (content outline I.B.1 / I.C.4).
Evidence for this item
- NCBI Bookshelf: DNA Replication — Lagging-strand synthesis produces Okazaki fragments joined by DNA ligase.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.B.1 / I.C.4
7. (MB-SCI-07)
A resistance gene carried on a plasmid moves from one bacterial cell to another through direct cell-to-cell contact using a pilus. What is this process called?
Show answer for question 7
Answer: C. Conjugation is transfer of DNA, often plasmids, through direct contact via a pilus. It is a major way resistance genes spread. A: transformation is uptake of free DNA from the environment. B: transduction uses a bacteriophage to carry DNA. D: transcription makes RNA from DNA; it isn't gene transfer.
Topic: Microbial genetics (content outline I.D.2).
Evidence for this item
- NCBI Bookshelf: bacterial conjugation — Conjugation transfers DNA between donor and recipient bacteria and is mediated by conjugative plasmid functions including pili.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline I.D.2
Molecular Techniques — Questions 8–17
8. (MB-TECH-01)
A DNA extract has an A260/A280 ratio of 1.5. Which interpretation is best?
Show answer for question 8
Answer: C. An A260/A280 ratio near 1.8 is typical for relatively pure DNA. A value of 1.5 is low, but the ratio is not specific for one contaminant: protein, residual phenol, and other extraction reagents can lower or distort it. A is inconsistent with the low ratio. B is not supported by this ratio alone. D cannot be concluded from the ratio; concentration limits are assessed from the instrument's absorbance range and full spectrum.
Topic: Nucleic acid quality (content outline III.B.1.b / II.A). Source: Thermo Fisher NanoDrop purity guidance.
Evidence for this item
- Thermo Fisher NanoDrop nucleic-acid purity guidance — Pure DNA is about A260/A280 1.8; low ratios can reflect protein, residual phenol, or other extraction-reagent contamination.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.B.1.b / II.A
9. (MB-TECH-02)
A DNA sample diluted 1:20 gives an A260 of 0.250. What is the concentration of the undiluted sample? (Use 50 µg/mL per A260 unit for double-stranded DNA.)
Show answer for question 9
Answer: C. 0.250 × 50 µg/mL × 20 = 250 µg/mL (the same as 250 ng/µL). A forgets the dilution factor (0.250 × 50). B and D come from misapplying the factor or the dilution. Carry the units through every step.
Topic: Concentration calculation (content outline III.B.2).
Evidence for this item
- Derived calculation from stated inputs — With the question-supplied conversion factor: 0.250 × 50 µg/mL × 20 = 250 µg/mL.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.B.2
10. (MB-TECH-03)
During agarose gel electrophoresis, DNA moves toward which electrode, and which fragments travel farthest?
Show answer for question 10
Answer: B. The phosphate backbone gives DNA a negative charge, so it moves toward the positive anode. The gel works like a sieve, so smaller fragments move faster and farther. The other choices reverse the direction, the size order, or both. ("Run to red" is the usual memory aid.)
Topic: Gel electrophoresis (content outline II.C.1.a).
Evidence for this item
- NCBI Bookshelf: DNA gel electrophoresis — DNA migrates toward the positive electrode and smaller fragments generally migrate farther through a gel matrix.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.C.1.a
11. (MB-TECH-04)
In a real-time PCR run, sample X has a Ct of 22 and sample Y has a Ct of 28 for the same target. Assuming 100% efficiency and equal input, how do they compare?
Show answer for question 11
Answer: B. A lower cycle threshold (Ct) means the signal crossed threshold sooner, so X started with more target. At 100% efficiency each cycle doubles the product: 2^(28 − 22) = 2^6 = 64. A reverses the direction and treats cycles as fold change. C uses the cycle difference as the fold change. D ignores what Ct measures.
Topic: Real-time PCR Ct (content outline II.D.2).
Evidence for this item
- MIQE 2.0 qPCR guidelines (2025) — qPCR quantification depends on amplification efficiency.
- Derived calculation — Under the question's ideal 100%-efficiency assumption, six cycles = 2^6 = 64-fold.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.D.2
12. (MB-TECH-05)
A hydrolysis (TaqMan) probe produces fluorescence during real-time PCR because:
Show answer for question 12
Answer: B. The probe binds between the primers. As the polymerase extends, its 5′→3′ exonuclease activity breaks the probe apart, freeing the reporter dye from the quencher. A describes a molecular beacon, which fluoresces when its hairpin opens on binding. C describes an intercalating dye such as SYBR Green. D describes paired FRET hybridization probes.
Topic: Probe chemistry (content outline II.C.5).
Evidence for this item
- PubMed: 5′ nuclease assays — Taq 5′→3′ nuclease activity cleaves an annealed fluorogenic probe during amplification.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.C.5
13. (MB-TECH-06)
A SYBR Green assay shows the expected melt peak at 84 °C in patient wells. An extra peak at 74 °C appears in several wells, including the no-template control. What is the most likely cause of the 74 °C peak?
Show answer for question 13
Answer: B. Intercalating dyes bind any double-stranded product, including short primer-dimers, which melt at lower temperatures. Its presence in the no-template control rules out a patient target. A would not appear in the no-template control. C lowers yield but doesn't create a consistent low-Tm product. D doesn't apply: SYBR assays don't use a probe.
Topic: Melt-curve analysis (content outline II.F.1).
Evidence for this item
- MIQE 2.0 qPCR guidelines (2025) — qPCR controls and melt analysis help identify nonspecific amplification and technical problems.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.F.1
14. (MB-TECH-07)
In Sanger sequencing, chain termination occurs because dideoxynucleotides (ddNTPs):
Show answer for question 14
Answer: A. Adding the next nucleotide requires a 3′-OH to form a phosphodiester bond. A ddNTP gets incorporated but has no 3′-OH, so the chain stops. That produces fragments ending at every base position. B: ddNTPs keep the triphosphate needed for incorporation. C and D aren't how termination works.
Topic: Sanger sequencing (content outline II.E.1).
Evidence for this item
- NCBI Bookshelf: Sanger sequencing — Dideoxynucleotides lack the 3′ hydroxyl required for continued DNA synthesis and terminate chains.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.E.1
15. (MB-TECH-08)
DNA is treated with sodium bisulfite before PCR and sequencing. At a CpG site confirmed as C in untreated DNA, the bisulfite-treated read shows T. What does this indicate about that cytosine in the original DNA?
Show answer for question 15
Answer: B. Bisulfite converts unmethylated cytosine to uracil, which PCR copies as thymine. Methylated cytosine is protected and remains read as C. Because untreated DNA already confirmed C at this site, a true C>T sequence variant has been ruled out for this question. Failed conversion would leave unmethylated cytosines reading as C rather than T.
Topic: Bisulfite conversion (content outline II.B.3). Source: Bisulfite sequencing methods.
Evidence for this item
- PubMed: bisulfite genomic sequencing — Bisulfite converts unmethylated cytosine to uracil, read after PCR as thymine; methylated cytosine is protected.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.B.3
16. (MB-TECH-09)
Which feature lets digital PCR report absolute target copy numbers without a standard curve?
Show answer for question 16
Answer: B. Digital PCR divides the reaction into thousands of tiny partitions so each holds zero, one, or a few copies. After amplification, the fraction of positive partitions is converted to copies with Poisson statistics, so no standard curve is needed. A: annealing temperature doesn't create absolute quantification. C: digital PCR reads endpoints, not cycle-1 fluorescence. D: it doesn't sequence.
Topic: Digital PCR (content outline II.D.2).
Evidence for this item
- dMIQE guidelines for digital PCR — Digital PCR uses partitioning and positive/negative partition counts with Poisson correction.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.D.2
17. (MB-TECH-10)
A lab must screen a tumor sample for variants across 50 genes, and some variants may be present in only a small fraction of tumor cells. Which approach fits best?
Show answer for question 17
Answer: B. NGS sequences many targets in parallel, and deep coverage (read depth) lets it detect variants present in a small fraction of reads. A: Sanger reads one amplicon per reaction and isn't designed to reliably detect low-fraction variants, so 50 genes would be slow and insensitive. C: a gel shows fragment size, not sequence. D: karyotyping detects large chromosomal changes, not point variants.
Topic: Sequencing method selection (content outline II.E.1–2).
Evidence for this item
- European LeukemiaNet 2025 recommendations — Targeted NGS provides sensitive variant detection and can detect lower-level variants that Sanger may miss.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline II.E.1–2
Laboratory Operations — Questions 18–22
18. (MB-LAB-01)
A qualitative PCR run shows amplification in the no-template control (NTC). What should the technologist do?
Show answer for question 18
Answer: C. A positive NTC means target or amplicon got into the reagents, so any positive in the run could be false. Hold results, find the source (reagents, work surfaces, amplicon carryover), decontaminate, and repeat. A risks reporting false positives. B has no valid basis. D leaves the contaminated run in place. The main preventive control is one-way workflow: pre-amplification to post-amplification, never back.
Topic: Contamination (content outline III.A).
Evidence for this item
- MIQE 2.0 qPCR guidelines (2025) — Negative/no-template controls are used to detect contamination and unexpected amplification requires investigation.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.A
19. (MB-LAB-02)
A patient sample is negative for the target, and its internal amplification control also fails. External controls worked. How should the result be handled?
Show answer for question 19
Answer: C. The internal control shows whether the sample and amplification process were adequate. If both target and internal control fail while external controls work, the negative target result is not valid. Follow the validated assay procedure to investigate inhibition or extraction failure and then repeat; depending on the assay, that may include re-extraction or an allowed dilution. A risks a false negative. B does not fit a target-negative result. D stops before the required troubleshooting/repeat step.
Topic: Internal controls (content outline III.B.4, III.B.6.a). Source: MIQE 2.0.
Evidence for this item
- MIQE 2.0 qPCR guidelines (2025) — Internal-control detection is essential to validate a negative result and can reveal inhibition or extraction failure.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.B.4, III.B.6.a
20. (MB-LAB-03)
Which anticoagulant is generally avoided for whole-blood specimens sent for PCR-based testing?
Show answer for question 20
Answer: B. Heparin can carry through extraction and inhibit DNA polymerases, so it's the classic tube to avoid. A: EDTA is the usual preferred tube. C: ACD is also commonly accepted. D is false. Always follow the specific assay's validated specimen requirements.
Topic: Specimen requirements (content outline III.B.1).
Evidence for this item
- PubMed: heparin inhibition of PCR — Heparin can inhibit PCR amplification.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.B.1
21. (MB-LAB-04)
A lab changes the specimen type on an FDA-cleared molecular assay from nasopharyngeal swab to saliva. Under CLIA, what must the lab do before reporting patient results?
Show answer for question 21
Answer: C. Using an FDA-cleared test outside its labeled claims, such as a new specimen type, makes it a modified test. CLIA (42 CFR 493.1253(b)(2)) then requires the lab to establish performance specifications: accuracy, precision, analytical sensitivity and specificity, reportable range, reference intervals, and others as applicable. A and B apply only to unmodified cleared tests (B is the 493.1253(b)(1) verification). D: the manufacturer can't satisfy the lab's CLIA obligation.
Topic: Test-system regulation (content outline III.C). Source: 42 CFR 493.1253(b)(1)–(2).
Evidence for this item
- 42 CFR 493.1253 — Modified FDA-cleared/approved tests require establishment of applicable performance specifications before patient-result reporting.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.C
22. (MB-LAB-05)
How do you make 500 mL of 1X TAE buffer from a 50X stock?
Show answer for question 22
Answer: A. Use C1V1 = C2V2: 50X × V1 = 1X × 500 mL, so V1 = 10 mL. Bring it to 500 mL total with 490 mL water. B gives 5X buffer. C gives 2.5X. D gives 0.1X.
Topic: Reagent preparation (content outline III.B.2).
Evidence for this item
- Derived dilution calculation — C1V1=C2V2 with 50X, 1X, 500 mL gives V1 = 10 mL concentrate and 490 mL diluent.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline III.B.2
Applications of Molecular Testing — Questions 23–32
23. (MB-APP-01)
A patient with chronic myeloid leukemia (CML) is on tyrosine kinase inhibitor therapy. Which test is used to monitor molecular response?
Show answer for question 23
Answer: B. CML is driven by the t(9;22) translocation, which creates the BCR::ABL1 fusion. Quantitative RT-PCR tracks transcript levels over time, and reporting on the International Scale (IS) makes results comparable across labs. A: JAK2 V617F is associated with other myeloproliferative neoplasms. C: t(15;17) defines acute promyelocytic leukemia. D: FLT3 matters in acute myeloid leukemia.
Topic: Leukemia monitoring (content outline IV.B.1). Source: MedlinePlus Genetics: ABL1.
Evidence for this item
- European LeukemiaNet 2025 CML recommendations — CML molecular monitoring uses quantitative BCR::ABL1 transcript measurement reported on the International Scale.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.B.1
24. (MB-APP-02)
A patient with recurrent venous clots tests positive for factor V Leiden. What causes the increased clotting risk?
Show answer for question 24
Answer: B. The factor V Leiden variant makes factor V resistant to inactivation by activated protein C (APC), so clotting stays active longer. A is a different inherited thrombophilia. C describes the prothrombin G20210A variant, also on the outline. D describes antiphospholipid syndrome, which isn't a germline variant.
Topic: Coagulopathy (content outline IV.C.2). Source: MedlinePlus Genetics: factor V Leiden thrombophilia.
Evidence for this item
- MedlinePlus Genetics: Factor V Leiden thrombophilia — Factor V Leiden makes factor V resistant to inactivation by activated protein C.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.C.2
25. (MB-APP-03)
FMR1 testing on a woman shows one allele with 30 CGG repeats and one with 75. How is the 75-repeat allele classified?
Show answer for question 25
Answer: C. Common FMR1 ranges: 44 or fewer normal, 45–54 intermediate, 55–200 premutation, more than 200 full mutation. A 75-repeat allele is a premutation, which can expand to a full mutation when a mother passes it to her children. A, B, and D use the wrong range.
Topic: Repeat disorders: FMR1 (content outline IV.C.3). Source: UW Laboratory Medicine: FMR1-Related Disorders.
Evidence for this item
- GeneReviews: FMR1 Disorders — FMR1 alleles of about 55–200 CGG repeats are premutations; full mutations are generally above 200.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.C.3
26. (MB-APP-04)
An adult's HTT testing shows 38 CAG repeats on one allele. How is this result interpreted?
Show answer for question 26
Answer: C. Common HTT ranges: 26 or fewer normal, 27–35 intermediate, 36–39 reduced penetrance, 40 or more full penetrance. At 38, disease is possible but not certain. A, B, and D place 38 in the wrong range.
Topic: Repeat disorders: HTT (content outline IV.C.3). Source: Huntington Society of Canada: Intermediate Alleles.
Evidence for this item
- GeneReviews: Huntington Disease — HTT 36–39 CAG repeats is the reduced-penetrance range; ≥40 is full penetrance.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.C.3
27. (MB-APP-05)
A child is suspected of having Prader-Willi syndrome. Which test can detect the abnormal maternal-only methylation pattern shared by the three major mechanisms: paternal deletion, maternal uniparental disomy, and an imprinting defect at 15q11-q13?
Show answer for question 27
Answer: B. DNA methylation analysis can establish the abnormal PWS imprinting pattern across the major mechanisms. However, methylation-only testing does not by itself identify whether the cause is a paternal deletion, maternal UPD, or an imprinting defect; additional dosage/segregation testing is used to determine the mechanism. Karyotype and FISH do not detect all three mechanisms, and sequencing a single gene misses the usual imprinting mechanisms.
Topic: Imprinting disorders (content outline IV.C.5). Source: EMQN/ACGS best-practice guideline.
Evidence for this item
- EMQN/ACGS best-practice guideline for PWS/AS testing — Methylation testing detects the abnormal imprinting pattern across major PWS mechanisms, but methylation-only testing does not distinguish the cause.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.C.5
28. (MB-APP-06)
A physician plans to start carbamazepine. Which result most strongly indicates increased risk of Stevens-Johnson syndrome or toxic epidermal necrolysis (SJS/TEN)?
Show answer for question 28
Answer: B. HLA-B*15:02 is strongly associated with carbamazepine-induced SJS/TEN; CPIC advises not to start carbamazepine in carriers who haven't taken it before. A: CYP2C19 status matters for clopidogrel activation. C: VKORC1 (with CYP2C9) affects warfarin dosing. D: HER2 amplification in a tumor predicts trastuzumab response. All four drugs are on the MB outline, so learn them as pairs.
Topic: Pharmacogenomics (content outline IV.D.3). Source: CPIC guideline for HLA genotype and carbamazepine/oxcarbazepine, 2017 update.
Evidence for this item
- CPIC HLA-carbamazepine/oxcarbazepine guideline — HLA-B*15:02 is associated with carbamazepine-induced SJS/TEN risk and informs therapy selection.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.D.3
29. (MB-APP-07)
After a hematopoietic stem cell transplant, the lab compares pre-transplant recipient, donor, and post-transplant samples to monitor engraftment. Which method is most commonly used?
Show answer for question 29
Answer: A. STR markers differ between donor and recipient. Informative alleles (present in one person but not the other) are measured in the post-transplant sample to estimate donor versus recipient percentage, called chimerism. B: ABO typing isn't used for quantitative chimerism. C: karyotyping only helps when donor and recipient differ in sex, and it's low resolution. D tracks infection, not engraftment.
Topic: Engraftment (content outline IV.D.2).
Evidence for this item
- Review: chimerism testing after allogeneic HSCT — STR-PCR remains a prevailing routine method for donor/recipient chimerism monitoring.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.D.2
30. (MB-APP-08)
A rapid PCR assay detects mecA in a Staphylococcus aureus isolate. What does this indicate?
Show answer for question 30
Answer: B. mecA encodes PBP2a, a penicillin-binding protein with low affinity for most beta-lactams. That's what defines MRSA. A: vancomycin resistance involves van genes. C: toxin genes are separate targets. D is the opposite of what mecA means.
Topic: Resistance testing (content outline IV.A.3).
Evidence for this item
- CDC: Laboratory Testing for MRSA — mecA-mediated resistance produces PBP2a and is associated with oxacillin/methicillin resistance.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.A.3
31. (MB-APP-09)
An EGFR variant is found in a lung tumor at a 30% variant allele fraction. The same variant is absent from an adequately tested matched blood sample, and sample identity is confirmed. What is the best interpretation?
Show answer for question 31
Answer: B. A variant present in tumor and absent from an adequately tested matched normal sample is most consistent with a somatic tumor variant. A is less likely because a constitutional germline variant is ordinarily detectable in appropriate normal tissue. C would also be expected in normal tissue. D is not supported when sample identity has been confirmed. In clinical practice, germline assessment still depends on the test design, tissue source, sensitivity, and the variant/gene context.
Topic: Somatic vs germline (content outline IV.B.2). Source: AMP/ASCO/CAP consensus.
Evidence for this item
- AMP/ASCO/CAP consensus on somatic variant interpretation — Matched-normal sequencing helps distinguish somatic from germline variants; VAF alone is insufficient.
- ACMG statement on germline variation in tumor testing — Normal-tissue interpretation can be complicated by clonal hematopoiesis and other postzygotic variants.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.B.2
32. (MB-APP-10)
A patient with metastatic non-small cell lung cancer has an EGFR exon 19 deletion. What is the main clinical use of this result?
Show answer for question 32
Answer: A. EGFR exon 19 deletions and exon 21 L858R are sensitizing mutations used to select EGFR-directed therapy such as osimertinib in advanced non-small cell lung cancer. Trastuzumab targets HER2, not a routine EGFR exon 19 deletion. A tumor EGFR driver does not by itself confirm a hereditary cancer syndrome, and warfarin dosing involves other pharmacogenes.
Topic: Oncology / therapy selection (content outline IV.B.2). Source: FDA: osimertinib-based therapy for EGFR-mutated NSCLC.
Evidence for this item
- FDA: osimertinib-based therapy for EGFR-mutated NSCLC (2024) — EGFR exon 19 deletion and exon 21 L858R are qualifying sensitizing mutations for osimertinib-based therapy in advanced NSCLC.
ASCP BOC MB Content Guideline (rev. Dec 5, 2025), outline IV.B.2
Score your practice set
Count your correct answers in each section.
| Section | Questions | Your score | What to do next |
|---|---|---|---|
| Molecular Science | 1–7 | __ / 7 | Fewer than 5 correct: start the plan in Week 1 as written |
| Molecular Techniques | 8–17 | __ / 10 | Fewer than 7 correct: give Weeks 1–2 extra time |
| Laboratory Operations | 18–22 | __ / 5 | Fewer than 4 correct: add the controls table and lab math to every review session |
| Applications | 23–32 | __ / 10 | Fewer than 7 correct: move Weeks 3–4 application sessions earlier |
Those cutoffs are our study rules of thumb, not ASCP readiness standards. A percentage on 32 questions is feedback on these questions only. ASCP reports a scaled score from a computer adaptive exam, and this set can't be converted to that score or used to predict a pass.
The fastest win: reread the explanation for every miss, write one sentence on why the right answer is right, and redo the question in a week without looking.
What's on the ASCP MB exam
| Item | Detail |
|---|---|
| Credential | Scientist in Molecular Biology, MB(ASCP). Older material may say "Technologist in Molecular Biology." MB(ASCPi) is a separate international pathway with different eligibility rules; this guide focuses on MB(ASCP). |
| Questions | 100 multiple choice, four options, one best answer |
| Time | 2 hours 30 minutes |
| Format | Computer adaptive testing (CAT) at Pearson testing centers |
| Navigation | You must answer each question to move on. You can flag questions and return if time remains. |
| Calculator | Onscreen calculator provided |
| Unscored items | Some field-test questions are mixed in and look the same as scored ones |
| Passing score | 400 on a 100–999 scaled score (not 40% correct) |
| Score report | Emailed notice within four business days once transcripts are processed; failing reports break the score down by content area |
| Application fee | $260, non-refundable |
Sources: MB content guideline, p. 1; ASCP About the Examination; ASCP MB credential page.
What CAT means for you: the first question sits near the passing standard. Get it right and the next is a little harder; miss it and the next is a little easier. There's no fixed number you need right. ASCP warns that missing questions on purpose to "get easier ones" backfires. Answer each one honestly and keep moving at about 90 seconds per question.
The four content areas
| Content area | Official weight | Topics in the outline | Practice questions |
|---|---|---|---|
| Molecular Techniques | 30–35% | Extraction; RFLP and bisulfite conversion; gel and capillary electrophoresis; probe hybridization and stringency; probe types (TaqMan, FRET, beacons, Scorpions); PCR design and variants (real-time, nested, multiplex, RT, allele-specific, digital); isothermal methods (Hybrid Capture, NASBA, TMA, LAMP); Sanger, NGS, pyrosequencing, RNA sequencing; bioinformatics (quality score, read depth); melt curves; arrays; MALDI-TOF | 8–17 |
| Applications of Molecular Testing | 30–35% | Infectious disease (qualitative panels, viral load, typing, resistance); leukemias, lymphomas, solid tumors, hereditary cancer; hemoglobinopathies, coagulopathies, repeat disorders, single-gene disorders, imprinting disorders, disease-associated HLA; identity testing; engraftment; pharmacogenomics | 23–32 |
| Molecular Science | 20–25% | Nucleic acid chemistry; replication, transcription, splicing, translation; chromosome, plasmid, phage, and mitochondrial structure; enzymes; human and microbial genetics | 1–7 |
| Laboratory Operations | 15–20% | Contamination; specimen and nucleic acid quality; reagent prep and calculations; validation and troubleshooting; QC and proficiency testing; instruments; ASR, RUO, IVD, LDP; CLIA, TJC, CAP, CMS, CLSI, FDA; competency; safety | 18–22 |
Two details from the guideline change how you study. Regulatory questions are based on U.S. sources (it names AABB, FDA, and CLIA), even for MB(ASCPi) candidates. And lab results and reference ranges appear in both conventional and SI units, so get comfortable reading both. Questions can be theoretical (apply knowledge, calculate, connect results to disease) or procedural (perform techniques, follow QA steps). Expect "what went wrong in this run?" more than "define this term."
Your 4-week ASCP MB study plan
This plan assumes six sessions a week, about 45–75 minutes each. It's our suggested starting point, not an ASCP requirement, and the right length depends on your background. Keep an error log: one line per miss with the topic and why you missed it.
| Week | Session | Focus |
|---|---|---|
| 1 | 1 | Take Questions 1–8; start your error log |
| 1 | 2 | Replication, transcription, splicing, translation, mutation types |
| 1 | 3 | Enzymes (polymerases, nucleases, reverse transcriptase, ligase), probes, hybridization, electrophoresis |
| 1 | 4 | Extraction, purification, nucleic acid quality and quantity; work the lab math below by hand |
| 1 | 5 | PCR basics: primer design, optimization, and the PCR family table |
| 1 | 6 | Redo every Week 1 miss without notes |
| 2 | 7 | Real-time PCR, digital PCR, melt curves, troubleshooting |
| 2 | 8 | Sanger sequencing and reading results |
| 2 | 9 | NGS workflow: library prep, sequencing, alignment, variant calling |
| 2 | 10 | NGS quality: quality scores, read depth, variant allele fraction |
| 2 | 11 | Isothermal amplification, arrays, epigenetic detection, MALDI-TOF |
| 2 | 12 | Take Questions 9–17; update your error log |
| 3 | 13 | Contamination control and the controls table |
| 3 | 14 | Validation, QC, proficiency testing, instruments |
| 3 | 15 | Test-system categories and regulations (ASR, RUO, IVD, LDP; CLIA, CAP, FDA) |
| 3 | 16 | Infectious disease: qualitative panels, viral load, resistance genes, typing |
| 3 | 17 | Oncology: CML and other translocations, clonality, solid-tumor markers, hereditary cancer |
| 3 | 18 | Take Questions 18–26; update your error log |
| 4 | 19 | Inherited disease: hemoglobinopathies, thrombophilias, repeat disorders, CF, hemochromatosis |
| 4 | 20 | Imprinting disorders, disease-associated HLA, identity testing, engraftment |
| 4 | 21 | Pharmacogenomics pairs |
| 4 | 22 | Mixed practice: method selection and troubleshooting |
| 4 | 23 | Take Questions 27–32; review the full error log |
| 4 | 24 | Final pass on your two weakest areas; check exam-day logistics |
Adjusting the plan:
- 2 weeks: do two sessions a day; keep every question and error-log session.
- 1 week: skip broad rereading. Do the full practice set, then spend the rest of the week on your two weakest sections and the reference tables.
- 6 weeks or more: keep this order, then add a second round of mixed questions and spaced review of your error log instead of adding new low-yield topics.
- Retaking: start with the content areas flagged on your score report and give them double sessions. Our ASCP exam results guide explains that report.
Molecular Techniques review
This is the biggest area, along with Applications. Learn each method by four questions: what it detects, what the result looks like, what can make it wrong, and which control catches the problem.
Worked example: studying real-time PCR that way
- What it detects: amplification measured cycle by cycle as fluorescence rises.
- What the result looks like: a Ct value. Within the same validated assay and comparable samples, lower Ct generally reflects more starting target; under the idealized 100%-efficiency assumption used in Question 11, each one-cycle difference is twofold.
- What can make it wrong: inhibitors (heparin, carryover from extraction), poor nucleic acid quality, contamination, primer-dimers with intercalating dyes (Question 13).
- Which control catches it: no-template control for contamination (Question 18), internal control for inhibition (Question 19), positive control for reagent or run failure, and standards for quantitative accuracy.
Apply the same four questions to every method below.
PCR family
| Method | What's different | Typical use |
|---|---|---|
| Conventional (end-point) PCR | Product detected after cycling, often on a gel | Presence/absence, fragment sizing |
| Reverse transcription PCR | cDNA made from RNA first | RNA viruses, fusion transcripts |
| Real-time PCR | Fluorescence read every cycle | Quantitation (viral load, BCR::ABL1), genotyping |
| Multiplex PCR | Several primer sets in one tube | Syndromic panels |
| Nested / hemi-nested PCR | A second round with internal primers | Higher sensitivity and specificity, at higher contamination risk |
| Allele-specific PCR | Primer 3′ end matches only one allele | Known point variants |
| Digital PCR | Sample split into partitions; positives counted | Absolute quantitation without a standard curve; low-level targets |
Probe and dye chemistries
| Chemistry | How signal appears |
|---|---|
| Intercalating dye (e.g., SYBR Green) | Binds any double-stranded DNA; needs a melt curve to confirm the product |
| Hydrolysis (TaqMan) probe | Polymerase 5′→3′ exonuclease cleaves the probe, freeing reporter from quencher |
| Molecular beacon | Hairpin opens when bound to target, separating reporter and quencher |
| FRET hybridization probes | Two probes bind next to each other; energy transfers only when both are bound |
| Scorpions | Primer and probe in one molecule; the probe binds within its own extended strand |
Isothermal and signal amplification
| Method | Key idea |
|---|---|
| TMA and NASBA | Constant temperature; reverse transcriptase plus RNA polymerase make RNA copies |
| LAMP | Constant temperature; several primers and a strand-displacing polymerase |
| Hybrid Capture | Signal amplification: RNA probes bind DNA targets, and antibodies detect the DNA:RNA hybrids |
Sequencing terms to know
- Sanger: ddNTP chain termination (Question 14). Strong for confirming one known region; not built for low-fraction variants.
- NGS: many fragments sequenced in parallel (Question 17).
- Quality score: the estimated chance a base call is wrong; higher is better.
- Read depth (coverage): how many reads cover a position. More depth gives more confidence, especially for low-fraction variants.
- Variant allele fraction: the share of reads carrying the variant. A heterozygous germline variant often appears near 50% in a diploid sample, but tumor VAF varies with tumor purity, copy number, clonality, and zygosity; VAF alone does not prove germline or somatic origin (Question 31).
- Bisulfite conversion: unmethylated C reads as T; methylated C stays C (Question 15).
Laboratory Operations review
When a control fails
| Result | What it means | What to do |
|---|---|---|
| No-template control amplifies | Contamination | Reject the run, find the source, decontaminate, repeat |
| Negative control positive | Contamination or carryover | Same as above |
| Positive control fails | Reagent, instrument, or run failure | Reject the run, troubleshoot, repeat |
| Internal control fails in one sample, target negative | Inhibition or extraction failure in that sample | Report invalid; re-extract or dilute and repeat |
| Internal control fails, target strongly positive | Target may have outcompeted the internal control | Follow the assay's validated interpretation rules for this pattern |
Prevent contamination with one-way workflow: reagent prep, then sample prep, then amplification and detection, with separate areas and equipment. Never carry amplified product back.
Test-system categories
| Category | Plain meaning |
|---|---|
| IVD | In vitro diagnostic product intended for clinical diagnostic use; follow cleared/approved labeling when applicable |
| ASR | Analyte-specific reagent: a specific reagent used to detect or quantify an analyte in a diagnostic application |
| RUO | Research use only: labeled “For Research Use Only. Not for use in diagnostic procedures.” |
| LDP | Laboratory-developed procedure/test system developed by the laboratory |
Under CLIA, before reporting patient results with an unmodified FDA-cleared or approved test, the laboratory verifies accuracy, precision, reportable range, and that the manufacturer's reference intervals are appropriate for its patient population. A modified FDA-cleared/approved test or an in-house test requires the laboratory to establish the applicable performance specifications before reporting results (42 CFR 493.1253; Question 21).
Lab math
Concentration from absorbance: A260 × conversion factor × dilution factor
| Nucleic acid | Conversion factor per 1.0 A260 |
|---|---|
| Double-stranded DNA | 50 µg/mL |
| RNA | 40 µg/mL |
| Single-stranded DNA and oligos (approximate) | 33 µg/mL |
Example: RNA diluted 1:10 reads A260 = 0.300. 0.300 × 40 × 10 = 120 µg/mL.
Purity ratios
| Ratio | Clean value (about) | A low value suggests |
|---|---|---|
| A260/A280 | 1.8 for DNA, 2.0 for RNA | Protein or phenol carryover |
| A260/A230 | About 2.0–2.2 | Salt, guanidine, phenol, or carbohydrate carryover |
Fold change from Ct: for the same target under the idealized 100%-efficiency assumption, fold difference = 2^ΔCt. About 3.3 cycles ≈ 10-fold; 6 cycles ≈ 64-fold. Lower efficiency changes that relationship.
Dilutions: C1V1 = C2V2. Example: to make 50 µL of 10 ng/µL DNA from a 250 ng/µL stock, V1 = (10 × 50) ÷ 250 = 2 µL stock plus 48 µL diluent.
Applications of Molecular Testing review
Repeat expansion disorders
| Disorder | Gene / repeat | Ranges |
|---|---|---|
| Fragile X–associated disorders | FMR1 / CGG | ≤44 normal · 45–54 intermediate · 55–200 premutation · >200 full mutation (source) |
| Huntington disease | HTT / CAG | ≤26 normal · 27–35 intermediate · 36–39 reduced penetrance · ≥40 full penetrance (source) |
Pharmacogenomic pairs named in the outline
| Drug | Marker | Why it matters |
|---|---|---|
| Carbamazepine | HLA-B*15:02 | Risk of SJS/TEN (CPIC) |
| Clopidogrel | CYP2C19 | Poor metabolizers activate less drug |
| Warfarin | CYP2C9 and VKORC1 | Dose requirement |
| Trastuzumab | ERBB2 (HER2) amplification in the tumor | Predicts response |
Oncology markers worth knowing cold
| Finding | Association |
|---|---|
| t(9;22), BCR::ABL1 | Chronic myeloid leukemia; monitored by quantitative RT-PCR on the International Scale (MedlinePlus) |
| t(15;17), PML::RARA | Acute promyelocytic leukemia |
| JAK2 V617F | Myeloproliferative neoplasms other than CML |
| EGFR exon 19 deletion or L858R | Non-small cell lung cancer; selects EGFR inhibitor therapy (FDA) |
| Clonal IG or TCR gene rearrangement | Supports a B-cell or T-cell lymphoid neoplasm |
Somatic or germline? A variant present in tumor and absent from an adequately tested matched normal sample is most consistent with a somatic change. A variant found in blood or another normal-tissue sample can support germline origin, but blood can also contain acquired clones such as clonal hematopoiesis. Clinical germline classification depends on the test design, tissue source, sensitivity, and variant context; tumor-only VAF is not enough by itself.
Inherited disease and identity
- Factor V Leiden: factor V resists inactivation by activated protein C (MedlinePlus). Prothrombin G20210A is the other common thrombophilia variant on the outline.
- Prader-Willi and Angelman syndromes: imprinting disorders at 15q11-q13. For Prader-Willi syndrome, methylation testing can detect the abnormal imprinting pattern across paternal deletion, maternal UPD, and imprinting-defect mechanisms, but methylation-only testing does not distinguish which mechanism caused the result (EMQN/ACGS guideline).
- Engraftment: STR analysis of informative alleles in recipient, donor, and post-transplant samples.
- Identity and parentage: STR profiles compared across samples; also used to resolve possible specimen mix-ups.
Molecular Science review
Most Molecular Science questions are really about the enzymes you use every day.
| Enzyme | What it does | Where you meet it in the lab |
|---|---|---|
| DNA polymerase | Adds nucleotides 5′→3′ | PCR, sequencing |
| Reverse transcriptase | Copies RNA into cDNA | RT-PCR, TMA, NASBA |
| Restriction endonuclease | Cuts DNA at a specific sequence | RFLP |
| Exonuclease | Removes nucleotides from an end | TaqMan signal, proofreading |
| DNA ligase | Seals breaks in the backbone | Okazaki fragments, library prep |
Also know how mutation types change a protein (Questions 3 and 5), why G–C content raises Tm (Question 1), how mitochondrial DNA is inherited (Question 4), and how bacteria share resistance genes (Question 7).
Final week checklist
- Redo every practice question you missed, without looking at the explanation first.
- Rebuild the repeat-disorder, PGx, and control-failure tables from memory.
- Work the lab-math examples once more, units included.
- Do one mixed set at about 90 seconds per question.
- Stop adding new resources; review your error log instead.
- Confirm your appointment, ID, and test center details in your ASCP Admission Notification and with Pearson.
Quick answers to common MB questions
Who can take MB(ASCP)? You need to meet one of four MB(ASCP) routes built around an existing qualifying ASCP certification, a NAACLS-accredited diagnostic molecular science program, or qualifying bachelor's/master's education plus molecular laboratory experience. Check your route on the ASCP MB credential page before you pay; it can't be changed after you submit. Step-by-step help: ASCP exam registration.
Can I retake it? Yes. You can test up to five times under one eligibility route, with a new application and fee each time. Your new eligibility period starts no earlier than 30 days after your previous exam date (ASCP After the Examination). Details: ASCP exam retake policy.
What should I read? ASCP publishes an MB suggested reading list, reviewed April 2026. ASCP says it's a partial list and doesn't imply endorsement. Buckingham's Molecular Diagnostics (4th ed., 2026) on that list follows the same four areas.
Is MB the same as SMB? No. SMB(ASCP), Specialist in Molecular Biology, is a separate, non-adaptive exam with its own eligibility. This guide covers MB only.
What happens after I pass? You keep the credential through ASCP's Credential Maintenance Program. See our ASCP CMP guide.
Sources
- ASCP BOC, Scientist in Molecular Biology – MB — credential name, eligibility routes, application fee
- ASCP BOC, MB(ASCP) and MB(ASCPi) Examination Content Guideline, revised December 5, 2025 — exam model (p. 1), content outline (pp. 2–3)
- ASCP BOC, About the Examination — item format, CAT, navigation, calculator, field-test items, scoring
- ASCP BOC, After the Examination — reapplication and attempt limits
- ASCP BOC, MB Suggested Reading
- 42 CFR 493.1253, Establishment and verification of performance specifications
- Thermo Fisher Scientific, NanoDrop nucleic-acid purity guidance
- Clinical Chemistry, MIQE 2.0 qPCR guidelines
- GeneReviews, FMR1 Disorders and Huntington Disease
- European LeukemiaNet, 2025 recommendations for chronic myeloid leukemia
- EMQN/ACGS, Best-practice guidelines for molecular analysis of Prader-Willi and Angelman syndromes
- CPIC, HLA genotype and carbamazepine/oxcarbazepine guideline
- CDC, Laboratory Testing for MRSA
- AMP/ASCO/CAP, Consensus recommendations for sequence variants in cancer
- U.S. FDA, Osimertinib-based therapy for EGFR-mutated non-small cell lung cancer
Last verified November 3, 2026: exam format, content guideline revision and weights, credential name, scoring, fee, retake rules, and reading list, against the ASCP BOC pages above.
Written by the Castleport Test Prep Editorial Team. Castleport Test Prep is an independent exam prep publisher and is not affiliated with, endorsed by, or approved by the American Society for Clinical Pathology (ASCP) or the ASCP Board of Certification. Exam and credential names are used for identification; trademarks belong to their respective owners. The practice questions on this page are original and unofficial, not real or recalled exam questions. Nothing here guarantees a passing score or employment.