MB ASCP Final Exam Questions and Answers Part 2 Latest Updates 2024 Graded A | Exams Nursing | Docsity (2024)

Download MB ASCP Final Exam Questions and Answers Part 2 Latest Updates 2024 Graded A and more Exams Nursing in PDF only on Docsity! 1 MB ASCP Final Exam Questions and Answers Part 2 Latest Updates 2024 Graded A+ What is the Beer-Lambert Law? - Answer the linear relationship between absorbance and concentration of an absorbing species. What is the Beer-Lambert Law equation? - Answer A = ebc A: absorbance e: molar absorptivity c: concentration b: path length DNA and RNA are absorbed at: - Answer 260nm Proteins are absorbed at: - Answer 280nm What is the melting temperature equation? - Answer Tm = (4 x # of GC pairs) + (2 x # of AT pairs) Dilutions equation - Answer V1C1 = V2C2 V1: volume of starting solution C1: concentration of starting solution V2: volume of new solution C2: concentration of new solution You received a 9ng DNA sample and have to make a 50ul dilution of this sample at 900pg. How many ul of stock do you need? - Answer 1ng = 1000pg convert 9ng to pg 9ng = 9000pg V1C1 = V2C2 9000pg x ? = 50ul x 900pg = 5ul of stock What is the concentration formula? - Answer Concentration = DNA/RNA constant x OD260 value x dilution factor 2 What does the PPV (positive predictive value) formula solve for? - Answer what is the chance that a person with a positive test truly has the disease? What is the PPV (positive predictive value) formula? - Answer TP / (TP+FP) x 100 TP: true positive FP: false positive What does the sensitivity formula solve for? - Answer the probability that a test will indicate 'disease' among those with disease What is analytical sensitivity based on? - Answer the limit of detection What is the sensitivity formula? - Answer TP / (TP+FN) x 100 TP: true positive FN: false negative What does the specificity test solve for? - Answer the fraction of those without disease who will have a negative result What is the specificity formula? - Answer TN / (FP+TN) x 100 TN: true negative FP: false positive What term best describes the ability to determine true value? - Answer accuracy What term best describes the reproducibility of independently determined test results? - Answer precision What is the A260/A280 ratio? - Answer It provides insight regarding the type of nucleic acid present as well as a rough indication of purity. Pure ds DNA has an A260/A280 ratio of 1.7-2.0 < 1.7 is protein contamination RNA ratio is around 2.1 A patient specimen is cut with RE and yields a 150 fragment + 210 fragment. The control is 360. How many cuts did the restriction enzyme make? - Answer 150 + 210 = 360 The restriction enzyme is making one cut. What analytical tests amplify the DNA target? - Answer qPCR (real time) Nested PCR Multiplex PCR LAMP (loop mediated isothermal amplification) This analytical test permits the identification of specific, amplified DNA fragments using analysis of their melting temperatures. The formation of amplicons is There is simultaneous amplification and real-time detection with high throughput and high sensitivity - Answer SDA (strand displacement amplification) What is necessary to make cDNA? - Answer reverse transcriptase What are the three biochemical activities of reverse transcription? - Answer RNA- dependent DNA polymerase Ribonuclease H DNA-dependent DNA polymerase --> all used to create ds cDNA from RNA Which techniques use isothermal amplification? - Answer NASBA, TMA, SDA, LAMP What analytical test uses treatment with Sodium bisulfite (NaHSO3) to convert unmethylated C to U - primers are then specific to strand w/ C and U? - Answer Methylation Specific PCR RFLP HD + enzyme BESS NIRCA Invader assay CCM Cycling Probe are all types of amplification - Answer cleavage What method uses visible light? - Answer pyrosequencing Where does the signal come from in pyrosequencing? - Answer luciferase conversion of luciferin to oxyluciferin What type of qPCR has two probes, one attached to a donor fluor (D) and one to an acceptor or reporter fluor (R)? This test hybridizes the 2 separate probes. When both hybridize in close proximity fluorescence will be emitted - Answer FRET (fluorescent resonance energy transfer) This analytical test is a mutation/ polymorphism detection technique using immobilized target and sequence specific probes with 1-2 mismatches. It uses Tm to see differences with probes that mismatch to those with perfect sequences (dot/slot blot). - Answer ASO (allele specific) This PCR method works by generating a signal at the annealing step (i.e. when the probe binds to its target) - Answer Molecular beacon MB (ASCP) Review & Exam Questions Part II Already Graded A+ This analytical test employs a set of primer pairs designed in such a way that they overlap, but only one will bind to the target in the presences of SNPs? - Answer ARMS (amplification refractory mutation system) What analytical assay uses fluorescent probes to detect DNA sequences on chromosomes? - Answer Fluorescent in situ hybridization (FISH) Explain how Taqman works - Answer TaqMan probes consist of a fluorophore covalently attached to the 5'-end of the oligonucleotide probe and a quencher at the 3'-end. What is the design of a molecular beacon? - Answer R-5'-sequence-3'-Q R: reporter Q: quencher What is the purpose of the MALDI TOFF matrix? - Answer It absorbs UV light and converts it to heat energy How does MALDI work? - Answer matrix-assisted laser desorption/ionization blasts surface with sample to produce positive ions that can be detected. Used for rapid ID of proteins and mass fingerprinting. How does MALDI TOFF work? - Answer the same as MALDI, but uses time of flight (TOF) to differentiate between ions. Lighter ions travel faster. What assay do you use to identify nucleic acid : protein combos? - Answer gel mobility shift assay Expression arrays are performed on: - Answer RNA MALDI methods separate ions by: - Answer mass and charge What does a DNA microarray test for? - Answer Gene expression. (DNA fingerprinting) Simultaneously assess the expression of multiple genes. Gene expression = look at mRNA in sample. cDNA sequences complementary to the mRNA of interest serve as a probe on the microarray-mRNA labeled and will hybridize and to cDNA. Light up read color with microarray reader. What is multiplex bead array? - Answer put many different probes with different labels - add patient specimen - flow cytometry Next Generation Sequencing uses what? - Answer Electrophoresis to separate bases by size or light detection at each cycle of synthesis Explain Illumina sequencing (NextGen) - Answer DNA molecules and primers are attached on a slide and amplified with polymerase to form DNA clusters. Four types of reversible terminator bases (RT-bases) are added an the non-incorporated MB (ASCP) Review & Exam Questions Part II Already Graded A+ nucleotides are washed away. Images are taken of the fluorescently labeled nucleotides as the sequence extends, then the dye, along with the terminal 3' blocker allowing for the next cycle to begin. Explain IonTorrent sequencing (NextGen) - Answer Uses standard sequencing chemistry, but a semiconductor based detection system. Based on the detection of hydrogen ions that are released during the polymerization of DNA as opposed to the optical methods. A microwell containing a template DNA is flooded with a single type of nucleotide (A,T,G,C) and if the nucleotide is complementary to the template it is incorporated into the growing strand of DNA. This causes the release of hydrogen ions that triggers the sensor. Explain SOLiD Sequencing (NextGen) - Answer Sequencing by ligation. A pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. The oligonucleotides are annealed and ligated, the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR and the resulting beads (each containing single copies of the same DNA) are deposited on the glass slide for sequencing. What are the steps in an NGS reaction? - Answer Divided into 4 samples (ddA, T, G, C) Label with radioactive/dye oligo at 3' end Mix with Taq, dNTPs, ddNTP and incubate run on gel --> frags will terminate at different lengths sequencing process involves fragmenting DNA/RNA into multiple pieces, adding adapters, sequencing the libraries, and reassembling them to form a genomic sequence. Why is next generation sequencing preferred over Sanger sequencing? - Answer higher depths of reads What reads for deletions and insertions in NGS sequencing? - Answer paired-ends What does depth refer to in NGS sequencing? - Answer how many times a unique position has been covered Next Generation Sequencing set-up requires what? - Answer library preparation and extensive bioinformatics analysis In NGS (next generation sequencing), what is coverage referring to? - Answer the number of times a base is read What are the steps of an organic isolation method? - Answer 1. Lyse 2. Add phenol/ chloroform > vortex/spin 3. Transfer aqueous layer (top) to new tube 4. Add chloroform:IAA (removes phenol) > vortex/spin MB (ASCP) Review & Exam Questions Part II Already Graded A+ The optimal annealing temperature for any given primer pair on a particular target can be calculated as follows: - Answer Ta Opt = 0.3 x(Tm of primer) + 0.7 x (Tm of product) - 25 What is the optimal annealing temperature? - Answer 5 degrees C below the Tm of primers How many base pairs are shorter probes? - Answer < 500 bp less specific possibility of random repeat in the genome sequence more sensitive to point mutations/polymorphism How many base pairs are longer probes? - Answer 500-5000 bp greater specificity decreased background less affected by point mutations/polymorphism Do you have to know the gene sequence in order to do DNA sequencing? - Answer Yes, in order to design primers You do NOT need to know the mutation What are the clinical uses for DNA sequencing? - Answer mutation detection confirm mutation by other method resistance testing HLA genotyping What does the incubation step do in hybridization? - Answer Allows formation of ds molecules In what order do you add the components of a PCR reaction? - Answer Master mix > positive control > patient samples > negative control What is an acceptable annealing temperature? What is an acceptable denaturing temperature? What is an acceptable extension temperature? - Answer Annealing temp: 50-70 Denaturing temp: 90-96 Extension temp: 68-75 The annealing temperature should not exceed what? - Answer The extension time What are the characteristics of melt curve analysis? - Answer it is the melting temperature of ds DNA depends on its base composition all PCR products for specific primer pair should have the same melting temperature and length MB (ASCP) Review & Exam Questions Part II Already Graded A+ Used for SNPs; Specimens with identical sequences should yield the same peak at the expected Tm and specimens containing different sequences will yield two or more peaks. In qPCR the is set above the background in the exponential phase - Answer threshold In Real-Time PCR, which phase is important for quantitation? - Answer exponential What part of a qPCR curve should you analyze? - Answer right as it starts to turn up, still in the linear phase What conditions increase PCR stringency? (associated DNAs have few mismatches) - Answer increase in temperature increase in probe length increase in probe complexity (high frequency of GC pairs) decrease in salt concentration decrease in MgCl2 concentration decrease in time decrease in formamide decrease in bovine serum decrease in probe concentration What conditions lower PCR stringency? (associated DNAs can have more mismatches) - Answer decrease in temperature decrease in probe length decrease in probe complexity (lower frequency of CG pairs) increase in salt concentration increase in MgCl2 concentration increase in time increase in formamide increase in bovine serum increase in probe concentration What will increase the specificity of a PCR reaction? - Answer using Nested PCR, using a high fidelity polymerase, or incorporating DMSO (dimethyl sulfoxide) or formamide There are weak loci for the multiplex PCR test. How do you get a good yield? - Answer increase the primer for the weak loci and at the same time decrease the primer for the loci that can be amplified. Balance between these amounts is important. MB (ASCP) Review & Exam Questions Part II Already Graded A+ Your PCR test is producing weak products / low yield. How do you change this? - Answer decrease the annealing temperature to the lowest possible increase the primer concentration to 0.4uM, template, and Taq polymerase Your PCR test is producing shorter, unspecific products. How do you change this? - Answer decrease KCl keep MgCl2 concentration at 1.5mM decrease the primers and template increase the annealing / extension time increase the annealing / extension temperature Your PCR test is producing longer, unspecific products. How do you change this? - Answer increase KCl keep MgCl2 concentration at 1.5mM decrease the annealing / extension time decrease the primers and template Your PCR test is producing weak, shorter products. How do you change this? - Answer Increase KCl Increase the amount of primers for weak loci Decrease the annealing time and temperature Decrease the extension time and temperature Your PCR test is producing weak, longer products. How do you change this? - Answer Decrease KCl Decrease the amount of primers for strong loci Increase extension time and temperature Increase annealing time Your two primers have very different melting temperatures but you can't change the locus. What can you do to improve PCR amplification? - Answer Increase the length of the primer with low Tm. Add a few bases to either end. After PCR, the amplification control has failed to yield a product. What should the technologist do? - Answer Check the original DNA preparation and if it is adequate repeat amplification. If not, re-isolate the nucleic acid. You run an assay with an internal control. The control works but one patients internal control does not. What do you do? - Answer repeat the patient Contamination of reagents with previous PCR products can be reduced by: - Answer Incorporating dU into the PCR products and then treating with UNG can reduce contamination of reagents with previous PCR products. What can cause primer dimers? - Answer annealing temp too low too much primer MB (ASCP) Review & Exam Questions Part II Already Graded A+ degradation loaded too much What are some causes of too many bands on a gel after PCR? - Answer primers not specific enough annealing temp too low too many cycles too much Mg++ What is one way you can increase the yield/quality of DNA/RNA after running gel? - Answer Do an EtOH nucleic acid precipitation Your gel electrophoresis is producing bands in the wrong place. How do you change this? - Answer Don't heat nucleic acids before running the gel Don't exceed 20 V/cm or 30 degrees C There are no bands evident for a DNA sample when ethidium bromide stained agarose gel is visualized on the transilluminator after running the gel for 15 minutes at 90 volts. The molecular weight ladder is not visible. The most likely reason for this finding is? - Answer The electric current was from anode to cathode. The correct orientation of a gel electrophoresis is from cathode to anode (black to red or negative to positive), if the gel is run from anode to cathode, the sample and molecular weight ladder would likely rn off the top of the gel. Evaluation of PCR product electrophoreses on a 0.8% agarose gel shown non- specific bands. The appropriate modification for the next PCR reaction is to: - Answer Reduction of primer concentration will result in an increased specificity of PCRs. What causes stutter anomaly (small peaks in engraftment)? - Answer strand slippage What percentage of the human genome consists of repeated sequences? - Answer 50% True or False. Proficiency testing is performed to assess the skills (competency of laboratory personnel performing molecular assays as well as the performance of the assay itself - Answer true You receive a paraffin embedded specimen for HPV typing, which method should you use? - Answer PCR the most common traditional diagnostic method for STIs and respiratory infection is: - Answer culture When performing proficiency testing it must be? - Answer run as a patient sample MB (ASCP) Review & Exam Questions Part II Already Graded A+ The best way to ensure no contamination or carryover in the lab is to use? - Answer Positive displacement pipettes How many SNPs (single nucleotide polymorphisms) are there? - Answer each individual has 11 million SNPs (10^7). So far, approximately 5 million SNPs have been identified in the human genome. NAACLS (CLSI) requires what to be in a procedure protocol as the absolute minimum - Answer Title, procedure, specimen requirements, reagents, interpretation, and signature of review MB (ASCP) Review & Exam Questions Part II Already Graded A+