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MB (ASCP) 2024
Consider the table below where a child and a possible father (PF) share the listed paternity indices for each locus listed (LOC-A1, LOC-B2, LOC- C3, LOC-D4). Locus Tested PF Child Paternity Index LOC-A1 3 2/3 2. LOC-B2 7/5 5 0. LOC-C3 15/17 9/17 5. LOC-D4 12 12 1. Based on the data presented in the table, what is the combined paternity index, CPI, from the loci tested: LOC-A1, LOC-B2, LOC-C3 and LOC-D4?
2.38 - ANS>12.42 ( CPI is calculated by multiplying all Paternity index tgt) Best way to avoid PCR contamination - ANS>Separate Pre-PCR (clean area) and post PCR (dirty area)
A260=1.4, A280=1.1. protein contamination or Rna contam or useable RNA - ANS>Protein contamination. A260/280 = 1. <1.6 = protein contamination, 1.7-2.0 = good DNA, 1.9-2.0 = good RNA Why is Fluorometry a better technique that Spectroscopy - ANS>Increased sensitivity and more importantly can accurately measure DNA concentration of a structurally intact double stranded DNA molecule Linkage Disequilibrium - ANS>LD is the non random association of alleles at 2 or more loci in a general population. When alleles are in LD, haplotypes do not occur at the expected frequencies. Specificity - ANS>Specificity = TN / (TN + FP) Limit of Detection (LOD) - ANS>lowest concentration of analyte that can be detected mRNA Sequence provided - Convert it into DNA - ANS>(Template/Anti- sense Strand) [Replace U with T] Correct order of HIV Sample Preparation Workflow - ANS>Plasma Preparation
Isothermal Method that utilizes Primes and Polymerase to Create Synthetic dsDNA - ANS>SDA (Strand Displacement Amplification) Polymerase used in Isothermal LAMP Procedure - ANS>BST (Bacillus Stearothermophilus) BRAF V600E Mutation is Associated with - ANS>Lynch Syndrome (Also Known as HNPCC) CAP Requires Discontinued SOP/Procedures to be stored for how long - ANS>At least 2 years from the date of discontinuation CAP Requires Laboratory Personnel (Technologist) Proficiency Testing - ANS>Once a Year Most Important Component of Clinical Laboratory Completed Validation Studies - ANS>Final Test Validation Summary Signed off by Laboratory Director STR Electropherogram provided and select the most informative loci - ANS>Two different alleles each was both recipient vs donor the most informative one STR makes use of which of the following - ANS>Variable Nucleotide
Experiment Performed (RFLP + PCR + Gel Electrophoresis): three bands observed in the mutant sequence as compared to the normal sequence
- ANS>Insertion of New Restriction Site Mutation in what gene decreases metabolism of Irinotecan Drug - ANS>UGT1A CYP2D6 Enzyme Metabolizes what drug - ANS>Codeine Determine Melting Temperature of a given DNA Sequence - ANS>2(A+T)
- 4(G+C) Specimen Collection for Testing of Cystic Fibrosis - ANS>Either 1 ml EDTA Lavender Tube/5 ml ACD Tube (Not Sure: I think I answered 1 ml EDTA, however the volume seems to be too low) Fish is used in - ANS>Prader Willi Syndrome Defects associated with Prader Willi Syndrome - ANS>Uniparental Disomy and Genomic Imprinting - Paternal Chromsome Deletion 15
How to run multiple unknow samples in a simple NGS Run - ANS>Barcoding the Samples Indexing is performed in NGS - ANS>To Barcode/Label/Tag DNA Sample of Interest Ideal Workflow Next Generation Sequencing (NGS) - ANS>Fragmentation-End Repair-Adapter Ligation What prevents/quenches the Fluorescent (Report Dye) Signal in a Taq Man Probe - ANS>G On 3' End A List of PCR Components for PCR Reaction Mix Provided (Determine which component is excess): - ANS>DNA Polymerase Concentration needs to be reduced to 2.5-3 units - Taq Polymerase Conc was provided as 100 Units as compared to other PCR Components How to Preferentially amplify short DNA Fragments - ANS>Increase KCL Concentration Degenerate Primer Sets - ANS>MultiPlex PCR
What PCR increases Specificity and sensitivity of a low copy number target in a patient sample - ANS>Nested PCR Pressure in pre-area and post-area PCR - ANS>Pre - Positive pressure Post - Negative pressure Bake Glassware to be used for RNA Isolation Procedure - ANS>250 C For 4 Hours (However Book States 4-6 Hours at 400 C) Isochromosomes - ANS>Long Arms Term for abnormal number of chromosomes in a cell. For example a human cell having 45 or 47 chromosomes instead of the usual 46. - ANS>Aneuploidy ( A cell with any number of complete chromosome sets is called a euploid cell.) Nucleic Acid Dyes that binds to minor groove of Double Helix DNA - ANS>SYBR Green Addition of Agarose in Gel - ANS>Slows the migration by 15% Polyacrylamide - ANS>Neurotoxin (Acrylamide Neurotoxin in Gel)
How many SNPS are there? - ANS>10^7 or 11 bilion How to create synthetic dsDNA? - ANS>SDA Culture of fibroblasts? - ANS>Trypsin and scrape Monitoring HIV circulating in patient's plasma using - ANS>NASBA HIV genome is RNA, so only NABSA would allow direct detection What is a metagenome? - ANS>sum total of all DNA sequenced from an environmental sample What gel to use for 200-300 base pairs? - ANS>0.5 agarose or 8% polyacrylamide (Pulse Field >25000bp PAGE 5-500 bp Agarose 200-30000bp) What does G574* mean? - ANS>Termination after the G How to store RNA for more than 6 months? - ANS>Ethanol -70C
How to check for HER2? - ANS>FISH What causes smearing in gel (high/low dye concentration, high low voltage) - ANS>High voltage Improperly prepared gel: If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear. If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel. Also DNA degradation. What is associated with multiple mutations and large deletions - ANS>DMD (Duchenne Muscular Dystrophy) _Dystrophin gene IQCP is most associated with - ANS>risk assessment Is Linkage Disequilibrium to nonrandom association of FAR or NEAR loci? - ANS>Near. (Linkage Disequilibrium occurs when 2 alleles are Close in the same chromosome. Linkage Equilibrium occurs when 2 alleles are Far apart in the same chromosome) Example of ISOTHERMAL SIGNAL amplification? Technique employs the signal amplification resulting from probe: target hybridization rather than by amplifying the target or the probe? - ANS>bDNA / bDNA analysis
Amplification method with polymerase creating a synthetic dsDNA - ANS>TBC Identical twins for organ donor what should you do? - ANS>continue with assay, tell supervisor, something about parents? Why does NGS use fluorescence over spectrometry? - ANS>Fluorescence is more sensitive because of the different ways of measuring absorbance and fluorescence. Light absorbance is measured as the difference in intensity between light passing through the reference and the sample. In fluorescence the intensity is measured directly, without comparison with a reference beam. Similarities between NGS and CE/Sanger - ANS>•DNA polymerase incorporates fluorescently labeled dNTPs into a DNA template •The newly incorporated nucleotides are identified by fluorophore excitation. MALDI-Tof - ANS>Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry (MS) is a common method used for quality control (QC) of oligonucleotides. MALDI-TOF instrumentation is suitable for use in high-throughput industrial QC settings and can resolve molecules in the size range of oligonucleotides
Heteroduplex - ANS>are dsDNA complexes - consisting of two partly mismatched polynucleotide strands derived from two different parent molecules. (look up Denaturing HPLC) Benefits of NGS Output - ANS>Reads are relatively short, but so much more is produced (100K-1 Billion) creating a huge amount of data for very little cost First time Real -Time PCR test perform, West Nile and HSV of CSF were negative after 10 days. Test repeated and HSV is now weak positive. Why? - ANS>Case series and studies have shown that HSV polymerase chain reaction (PCR) can be falsely negative, especially among children and early in the disease course. If testing first LP is negative and herpes simplex encephalitis (HSE) is still of concern, a second LP should be repeated within 3-7 days with CSF sent for HSV PCR. Perfect PCR conditions, 5ng/mL DNA used produces faint read, how to increase the read? - ANS>First check your programming for each step of PCR cycle as the faint bands are due to several reasons like insufficient number of your cycles, low extension time, low annealing time, increased annealing temperature, decreased denaturing temperature, high or low denaturation time. How a TaqMan probe should look - ANS>Flourophore / Quencher
● HBV
● HCV
● HPV - ANS>HPV
Genetic imprinting - ANS>Occurs when the expression of a gene has different effects depending on whether the mother or the father passed on the gene Epigenetics - ANS>the study of environmental influences on gene expression that occur without a DNA change Warfarin (Coumadin) - ANS>VKorC1, CYP2C9 (Treats thrombosis (Factor V Leiden, Prothrombin)) Palindrome - ANS>a word or phrase that reads the same backward as forward Writing primer forward/reverse sequences - ANS>Forward: Start writing complimentary strand for 3'-5' strand (about 20nt and end at G-C) => got forward primer 5'-3' Reverse: 20 complimentary nt to 5'-3' strand from 3'- 5' direction. Then rewrite the primer to 5' to 3'
RFLP - ANS>a variation in the length of restriction fragments produced by a given restriction enzyme in a sample of DNA. Such variation is used in forensic investigations and to map hereditary disease. NGS steps - ANS>1. Prepare genomic DNA sample. Randomly fragment DNA and ligate adapters onto end of DNA
- Attach DNA to surface. Bind single stranded fragments randomly to the inside surface of the flow cell channel.
- Bridge Amplification. Added unlabeled nt & enzyme to initiate solid-phase bridge amplification.
- Fragments become double stranded. Enzyme incorporated nt build ds briges
- Denature ds molecular denaturation leaves ss templates.
- Complete Amplification. Several million dense clusters of ds DNA are generated in each channel of the flow cell.
- First chemistry cycle: determine first base. Add 4 labeled reversible terminators, primers, and DNA polymerase enzyme to flow cell.
- Image first base: capture image of fluorescence after excitation and record identity of each cluster. Excitation removes allylyl group allowing next nt to bind 9: repeat 7 and 8
A technologist uses the spectrophotometer to quantify the amount of DNA extracted from a blood specimen diluted 1:30. The absorbance reading at 260 nm was found to be 2.545. The concentration of DNA in this extract is: A. 76.35 micrograms/mL B. 3817.5 micrograms/mL C. 381.75 micrograms/mL D. 3054.0 micrograms/mL - ANS>B (2.545 * 30 * 50 ) 50 for DNA 40 for RNA This enzyme is crucial in the making RNA from DNA: A. DNA Polymerase B. RNA Polymerase C. Helicase D. Primase - ANS>B The nitrogenous base in a nucleotide is expected to be found on which position of the sugar? A. C1' B. C2' C. C3'
D. C5' - ANS>A
Its discovery shed light on why there is simultaneous, though not continuous, synthesis of DNA on both leading and lagging strands of DNA: A. Klenow fragment of DNA polymerase B. Okazaki fragments C. Sanger fragments D. RNA fragments - ANS>B The process of making RNA is termed: A. Replication B. Translation C. Transcription D. Polymerase chain reaction - ANS>C The enzyme that primes DNA synthesis is: A. DNA polymerase B. Exonuclease C. DNA ligase D. DNA primase - ANS>D
