MB (ASCP) Review & Exam Questions Part II Questions and answers 2023

MB (ASCP) Review & Exam Questions Part II Questions and answers 2023 What is the Beer-Lambert Law? the linear relationship between absorbance and concentration of an absorbing species. What is the Beer-Lambert Law equation? A = ebc A: absorbance e: molar absorptivity c: concentration b: path length DNA and RNA are absorbed at: 260nm Proteins are absorbed at: 280nm What is the melting temperature equation? Tm = (4 x # of GC pairs) + (2 x # of AT pairs) Dilutions equation 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? 1ng = 1000pg convert 9ng to pg 9ng = 9000pg V1C1 = V2C2 9000pg x ? = 50ul x 900pg = 5ul of stock What is the concentration formula? Concentration = DNA/RNA constant x OD260 value x dilution factor What does the PPV (positive predictive value) formula solve for? what is the chance that a person with a positive test truly has the disease? What is the PPV (positive predictive value) formula? TP / (TP+FP) x 100 TP: true positive FP: false positive What does the sensitivity formula solve for? the probability that a test will indicate 'disease' among those with disease What is analytical sensitivity based on? the limit of detection What is the sensitivity formula? TP / (TP+FN) x 100 TP: true positive FN: false negative What does the specificity test solve for? the fraction of those without disease who will have a negative result What is the specificity formula? TN / (FP+TN) x 100 TN: true negative FP: false positive What term best describes the ability to determine true value? accuracy What term best describes the reproducibility of independently determined test results? precision What is the A260/A280 ratio? 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? 150 + 210 = 360 The restriction enzyme is making one cut. What analytical tests amplify the DNA target? 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 measured in real time during the exponential phase. EtBr or SyBr green intercalates and fluoresces qPCR (real time) This analytical test amplifies the same target in two PCR reactions and uses the product of round 1 as the template for round 2. So there are 2 pairs of primers and 2 successive reactions. Basically you pre-amplify a broad region and then amplify a specific region. This test is good to use if you have low amounts of target because it increases specificity/sensitivity. Nested PCR This analytical test has multiple pairs of primers to test multiple things simultaneously, makes multiple products, and is good for typing/identification. The annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR This analytical test has one tube and one constant temperature, makes more amplicons than PCR, uses four different primers to identify six distinct regions on a target gene (creates overhangs) , has high specificity, and is a low cost alternative. Uses Bst polymerase. Can be visualized by eye or photometer. LAMP (loop mediated isothermal amplification) What analytical tests amplify the RNA target? RT PCR (reverse transcriptase) TMA (transcription mediated amplification) NASBA (nucleic acid sequence based) This analytical test makes cDNA from RNA using reverse transcriptase. It is good for viruses and the DNA yield is low but specific. Can be used to quantify messenger RNA (mRNA). RT PCR (reverse transcriptase) This analytical test is a version of TAS, is isothermal, uses reverse transcriptase, and can simultaneously detect multiple pathogenic organisms in a single tube. There is a replication of DNA or RNA target through an intermediate RNA hybrid. Uses RNase to degrade RNA. TMA (transcription mediated amplification) This analytical test makes cDNA from RNA at a constant temperature, tests viral load, produces multiple copies of ss RNA, and is a two step process that uses RNase H to destroy the RNA in RNA-DNA. It uses 3 primers. Uses a single mixture. Uses T7-RNA Po. The primers and template are in contact at 22°–25°C, a condition of very low stringency NASBA (nucleic acid sequence based) What analytical tests amplify the signal? bDNA (branched DNA) Cleavase (invader) Hybrid capture MLPA (multiplex ligated dependent probe amplification) This analytical test uses a series of short oligomer probes to capture the target. Extender probes bind to the target and then to reporter molecules, loading the target nucleic acid with signal. Alkaline phosphatase is used to generate chemiluminescence. This test can detect diseases that have different isolates because you can test multiple probes that detect slightly different sequences. The ELISA of DNA. bDNA (branched DNA) What molecular technique can use RNA or DNA as the starting substrate? bDNA This analytical test focuses on mutation/polymorphism detection using sequence specific probes and a cleavase enzyme that binds at target. The 5' end of the signal probe overlaps with the invader probe and cleavase cleaves the signal probe. Fluorescence is generated. Cleavase (invader) This analytical test is an ELISA like assay using antibodies to an RNA:DNA hybrid formed by the hybridization of target RNA with a DNA probe. The hybrid is recognized by fixed antibodies and detected with alkaline phosphatase (antibodies bind and sandwich hybrid from other side). It is easier and cheaper than PCR. Alkaline phosphatase generates chemiluminescence. Hybrid capture This analytical test is multiplex PCR mixed with LCR (ligase chain reaction). The probes are two separate oligonucleotides, each containing one of the PCR primer sequences. They hybridize to immediately adjacent target sequences and are ligated together to produce a signal. It detects mutations and DNA methylation. MLPA (multiplex ligated dependent probe amplification) What analytical tests amplify the probe? LCR (ligase chain reaction) Q-beta replicase SDA (strand displacement amplification) This analytical test uses four synthetic oligonucleotide probes to anneal at specific target sites on the cryptic plasmid. Primers bind adjacent and ligase binds them together/seals nick. These primers now serve as templates for annealing and ligation of additional primers. This test is good for point mutations and is more specific than PCR. LCR (ligase chain reaction) This analytical test uses an RNA dependent RNA polymerase. The assay amplifies the probe signal and the probe RNA concentration increases if the target to be detected is present. The target (ssDNA/RNA) is added to tube w/ reporter probe. Target:reporter hybridize to capture probes The complex is then hybridized to capture probe with magnetic bead The complex is bound to well and washed The target:reporter is released QBpol is added The probe is amplified and detected via colorimetric or fluorogenic methods Target can be either DNA or RNA. Q-beta replicase This analytical test amplifies using nick translation and strand displacement. Nicking --> extension --> displacement. Uses two primers (one has a restriction site). Nuclease nicks primer and allows other primer to keep amplifying and displacing 1st strand. There is simultaneous amplification and real-time detection with high throughput and high sensitivity SDA (strand displacement amplification) What is necessary to make cDNA? reverse transcriptase What are the three biochemical activities of reverse transcription? RNA-dependent DNA polymerase Ribonuclease H DNA-dependent DNA polymerase --> all used to create ds cDNA from RNA Which techniques use isothermal amplification? 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? Methylation Specific PCR RFLP HD + enzyme BESS NIRCA Invader assay CCM Cycling Probe are all types of _____ amplification cleavage What method uses visible light? pyrosequencing Where does the signal come from in pyrosequencing? 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 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). ASO (allele specific) This PCR method works by generating a signal at the annealing step (i.e. when the probe binds to its target) Molecular beacon 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? ARMS (amplification refractory mutation system) What analytical assay uses fluorescent probes to detect DNA sequences on chromosomes? Fluorescent in situ hybridization (FISH) Explain how Taqman works 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? R-5'-sequence-3'-Q R: reporter Q: quencher What is the purpose of the MALDI TOFF matrix? It absorbs UV light and converts it to heat energy How does MALDI work? 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? 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? gel mobility shift assay Expression arrays are performed on: RNA MALDI methods separate ions by: mass and charge What does a DNA microarray test for? 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? put many different probes with different labels - add patient specimen - flow cytometry Next Generation Sequencing uses what? Electrophoresis to separate bases by size or light detection at each cycle of synthesis Explain Illumina sequencing (NextGen) 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 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) 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) 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.