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ASCP Exam Questions and Answers 2024
Which is required to generate cDNA A. DNA polymerase B. RNase H C. Restriction Enzyme D. Helicase - correct answer B What is the function of Taq Polymerase - correct answer recognize dsDNA You have a 50 μL primer stock at 9pM/μl and want dilute the stock to 900nM. How much stock should you add(what is the dilution factor) - correct answer 2 What gene causes hypersensitivity to Irinotecan? - correct answer UGT1A Which allele causes hypersensitivity to Carbamazepine? - correct answer HLA- b* What gives MLPA its specificity - correct answer Stuffer Sequence How is copy number determined? - correct answer MLPA What is the gene that cause resistance to Vancomycin - correct answer vanA Which assay is FISH best used for - correct answer Prader Willi RNA polymerase II: where is it and what does it do - correct answer Eukaryotic: It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA. (transcribes mRNA) Hazard that is blue and is level 4 what does it mean - correct answer big health hazard Dr calls wanting test results but results have not been signed out by a medical director what do you do - correct answer don't give out the result until it has been signed out my the MD What trinucleotide repeat is involved in Fragile X? - correct answer CGG In Fragile X, what is considered a full mutation expansion? - correct answer 200+ What method is used to detect Fragile X? - correct answer Southern blot (now PCR
/mPCR)
When doing a multiplex you observe faint bands. What do you do - correct answer Adjust the annealing temp
What do you do when you want to optimize a PCR reaction that generates short PCR fragments? - correct answer Adjust annealing temperature Which is caused by a T-cell rearrangement
- Mantle Cell
- Burkits
- Hodkins Lymphoma
- Sezary - correct answer Sezary What protects DNA as it is being replicated - correct answer Single Stranded Binding Protein What opens the DNA double helix up - correct answer Helicase What unwinds spiral DNA - correct answer topoisomerase What does HCV assays target - correct answer 5' UTR region How does TaqMan work? - correct answer fluorescence is generated when the quencher is hydrolyzed Which is the best order to limit contamination
- Mastermix/ patient/ positive/ negative
- Patient/ Mastermix/ negative/positive
- Matermix/ negative/ patient/ positive
- Mastermix/ positive/ patient/ negative - correct answer Matermix/ negative/ patient/ positive How does Luminex work - correct answer Tests one patient with many beads How do histones bind to DNA - correct answer ionic charges Histones are associated with what amino acids - correct answer Lysine & Arginine Methylation and Histone Modification are examples of what - correct answer epigenetics Variations in Maternal & Paternal DNA mean what - correct answer imprinting HLA typing over serology has the advantage of detecting what - correct answer null mutations Doctor suspects that a patient has clonality you run a PCR and gel and the patient sample, the positive control, and the negative control all show a band. What do you do - correct answer repeat testing
- Perform Southern Blot - correct answer Repeat RT-PCR What is the equation for PPV(positive predictive value) - correct answer PPV=TP/ (TP+FP) (#true positives)/(# true positives + # false positives) What can you expect from a multiplex PCR - correct answer you will need to increase the amount of magnesium Translation t(14, 18) - correct answer Follicular Lymphoma DNA Methylation will result in which of the following:
- inhibition of transcription
- inhibition of translation
- gene mutation - correct answer inhibition of transcription (protein inactivation) Real-Time PCR is better as a quantitative then conventional tests based on which of the following?:
- Gives you greater dynamic range
- Can monitor each cycle better
- Can use CT to determine results - correct answer Can use CT to determine results For PCR, I get (many) longer unspecific products. What can I do? - correct answer Decrease annealing time Increase annealing temperature Decrease extension time Decrease extension temperature to 62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM. Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases) and change the primer(s) Combine some/all of the above For PCR, I get (many) shorter unspecific products. What can I do? - correct answer Increase annealing temperature Increase annealing time Increase extension time Increase extension temperature to 74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x,
but keep MgCl2 concentration at 1.5-2mM
the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer.
I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them?. How? - correct answer Very likely, yes. Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2). Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4º C. If some of the loci are weak or not amplified, troubleshoot. How many loci can I amplify in multiplex PCR at the same time? - correct answer Difficult to say. The author has routinely amplified from 2 to 14 loci. Literature describes up to 25 loci or so. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them? - correct answer The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used !!. Check primer sequences for primer-primer interactions Short PCR products in my multiplex reaction are weak. How can I improve their yield? - correct answer Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5- 2mM Decrease denaturing time Decrease annealing time and temperature Decrease extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the "strong" loci. Add adjuvants. Best, use BSA (0.1 to 0. μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above Longer PCR products in my multiplex reaction are weak. How can I improve their yield?
- correct answer Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Increase denaturing time Increase annealing time Decrease annealing temperature Increase extension time and temperature Increase amount of primers for the "weak"
concentration) DMSO or glycerol Combine some/all of the above All products in my multiplex reaction are weak. How can I improve the yield? - correct answer Decrease annealing temperature in small steps (2º C) Decrease extension temperature to 62- 68º C Increase extension time Increase template concentration Increase overall primer concentration Adjust Taq polymerase concentration Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Add adjuvants. Best, use BSA (0.1 to 0. μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol Unspecific products appear in my multiplex reaction. Can I get rid of them somehow? - correct answer If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM If short: decrease buffer concentration to 0.7- 0.9x, but keep MgCl2 concentration at 1.5- 2mM Gradually increase the annealing temperature Decrease amount of template Decrease amount of primer Decrease amount of enzyme Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Add adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol If nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific products in the multiplex reaction. Combine some/all of the above Note: primer-primer interactions in multiplex PCR are usually translated
into lack of some amplification products rather than the appearance of unspecific products After an amplification procedure followed by gel electrophoresis, you took a photo of your gel. You notice consistent bands in all your gel lanes. However, you also see what appears to be a faint but noticeable band in your reagent blank lane. What is your best explanation for the reagent blank band?
answer 1. gene locus
- disease-causing variants (mutations) tested 3. genotype-phenotype correlations
- analytic sensitivity and specificity When is correlating molecular results with cytogenetic evaluation useful? - correct answer for translocation testing such as Bcr./abl in malignancies What should ideally occur when studying diagnostic accuracy of a test? - correct answer all patients undergo the index test and are evaluated using a reference standard A 49-year-old younger brother of your patient has been given a diagnosis of HH. DNA testing revealed homozygosity for the C282Y allele. For this reason, you patient requested DNA testing. Homozygosity for the C282Y allele was observed. Your patient has no clinical findings associated with hemochromatosis. Explain. - correct answer Incomplete penetrance. What are key characteristics of DNA linkage studies? - correct answer -they are useful for large genes when no common mutations exist
- they require participation from multiple family members
- they rely upon polymorphic DNA markers in close proximity to the disease gene
- they can yield erroneous results as a result of genetic recombination In which of the following diseases is homozygosity for two mutant alleles not associated with a more severe clinical phenotype: -achondroplasia -charcot-marie-tooth -huntington -thrombophilia - correct answer huntington Expansion of the CGG repeats in an FMR1 premutation allele to a full mutation allele: a. occurs during DNA replication b. is less likely to occur with a CGG repeat length of 79 as compared with 59 c. is dependent on the sex of the fetus d. is more likely to occur during male gametogenesis. - correct answer a When monitoring HIV-1 infected patients with viral-load assays, what change in viral load represents a biologically relevant change in viral replicatione - correct answer 0.5log(10) (threefold) The current FDA-cleared HIV-1 genotyping assays detect mutations that confer resistance to all of the following classes of drugs EXCEPT: a. nucleoside reverse transcriptase inhibitors b. nonnucleoside reverse transcriptase inhibitors c. protease inhibitors d. fusion inhibitors - correct answer d. fusion inhibitors
The Epstein-Barr virus is frequently identified in what type of tumor cells? - correct answer tumor cells of endemic Burkitt lymphoma Label each of the following mutations: a) p.Thr603del b) p.L439X c) c.348-2A>C d) c.745_746insTCGA e) p.Met325Lys - correct answer a) in-frame deletion b) nonsense c) splice site d) frameshift e) missense What are the steps of MLPA - correct answer 1. Probe Hybridization 2. Ligation
- PCR/Amplification
- Capillary Electrophoresis Indicate whether or not you would recommend anti-EGFR therapy in each of the following patients with lung adenocarcinoma: EGFR mutation detected: -D761Y -L858R -E746-A -T790M - correct answer YES: L585R/E746-A What type of testing would you recommend to a doctor who is considering treating a stage IV colorectal cancer patient with Cetuzimab? Why? - correct answer KRAS/NRAS testing by pyrosequencing since mutations in these genes cause Cetuzimab to be ineffective. It is unwise to start a patient on this drug prior to testing as anti-EGFR therapies such as Cetuximab can be associated with toxicity and are also very expensive for the patient. It would be BAD to give a toxic and expensive drug to a patient who would not respond to the therapy. Which end of the sequenced DNA is found at the bottom of the sequencing gel? - correct answer 5 ' When two sequences are found to be the same, they have what? - correct answer Identitiy Hereditary hemochromatosis is characterized by excessive storage of iron in the liver, skin, pancreas, heart, joints, and testes. Symptoms may include liver cirrhosis, progressive increase in skin pigmentation, diabetes mellitus, congenital heart failure
and/or arrhythmias, arthritis, and hypogonadism. This is an example of what? - correct answer Pleiotropy Less than 2% of individuals who are compound heterozygous for the C282Y and H63D mutations in HFE will develop clinical symptoms of hereditary hemochromatosis. This is an example of what? - correct answer incomplete penetrance Explain how and why the hemophilia A testing scheme differs for patients with severe disease vs. those with moderate disease - correct answer -severe: most commonly caused by inversions (47%) Testing= inversion--> sequencing--> del/dup -moderate: most commonly caused by missense mutations Testing= sequencing--> del/dup What is the minimum log difference that is significant with HepC? - correct answer 1.0 log Where is HLA-Dr located? - correct answer Exon 2, beta What are the 4 major stages of pyrosequencing? - correct answer 1. PCR 2. Clean-up
- Sequencing
- Analysis What does high quality DNA look like on a gel - correct answer stays at the top of the well (shouldn't smear) What is the number of bp in a human haploid cell? - correct answer 3. billion (most cells diploid which would be 6.6billion) What are the three stop codons? - correct answer UAA(TAA), UAG(TAG), UGA(TGA) What are you worries about is plasma is spun down from 7 say old whole blood in regards to CMV viral loads? - correct answer Possible PCR inhibition from hemolysis of RBCs What is the Methicillin resistance mechanism(such as in MRSA with the mecA gene)? - correct answer Enables cell wall synthesis to continue in the presence of antibiotics In real time PCR curves, where is the crossing threshold measured? - correct answer Exponential phase What is the advantage of real-time PCR with quantitative assays over some other methods? - correct answer can get exact CT and then use a std. curve to quantify What translocation causes acute promyeloid luekemia? - correct answer
