ASCP Molecular Biology Certification Exam Questions and Answers 100% Pass

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ASCP Molecular Biology Certification Exam Questions and Answers 100% Pass Pyrimidine One carbon ring Cytosine, Thymine, Uracil What is the function of mRNA? Carries genetic info out of nucleus ... Transcript translated to protein What is the function of tRNA? Carries aa to ribosome Anticodon pairs with codon on mRNA strand What is the function of rRNA? part of ribosome structure most abundant RNA coordinated coupling of tRNA to mRNA codons Feedback inhibition Product of pathway is noncompetitive inhibitor Binds to allosteric site to slow down rxn b/c too much product Endonucleases (Prok) Restriction enzymes Cleaves phoshpodiester bonds w/i poly-nt chain Recognition site is palindromic sequence Types I-V ORI sites nt sequence where replication is initiated Topoisomerase I Induces ss breaks Remove DNA supercoils during TXN and DNA replication; for strand breakage during recombination; for chr condensation; and to disentangle intertwined DNA during mitosis topoisomerase II cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strands Gyrase (topoisomerase II) Unwinds supercoiling caused by unwinding at the rep fork by introducing DSBs Helicase Breaks H-bonds of double helix at the replication fork single-strand DNA binding proteins (SSBPs) Binds ssDNA and prevents it from re-annealing during TXN, replication, repair, and recombination Telomeres Repeat sequence (TTAGGG) at the ends of chr, protect chr from degradation RNA polymerase DNA dependent RNApol Transcribes DNA template to RNA (3'-->5'; anti-parallel) Splicesomes Complex of snRNPs Removes introns from pre-mRNA and splices exons together Enhancers Short regions of DNA that bind proteins (TXN factors) that enhance TXN of a gene Reverse transcriptase enzyme that transcribes RNA to cDNA (lacks introns) RNA --> RNA:DNA --> cDNA (dsDNA) Open Reading Frame (ORF) sections of DNA that begin with start codons and end with stop codons DNA: 5' --> 3' transcription: 3' --> 5' DNA --> RNA (promoter) translation: 5' --> 3' mRNA Spectrophotometer Measures amount of light absorbed Quantitative measurement of [DNA/RNA] At what wavelength does DNA and RNA absorb? 260 nm At what wavelength does protein absorb? 280 nm Organic isolation method 1. Lyse 2. Add phenol/ chloroform > vortex/spin 3. Transfer aqueous layer (top) to new tube 4. Add chloroform:IAA (removes phenol) > vortex/spin 5. Transfer aqueous layer to new tube 6. Add NaOAc and EtOH > vortex/spin 7. Decant 8. Resuspend What does the incubation step in hybridization do? Allows formation of ds molecules Formamide acts as a __________ in a hybridization. denaturing agent Line Probe Assay (LiPA) reverse hybridization assay using sequence-specific oligonucleotide probes (reverse SSOP) multi-parameter testing --> single strip Single-Stranded Conformational Polymorphism Ananlysis (SSCP) Used for known gene, unknown mut Mutation screening Short PCR products form 3D conformation when cooled --> muts have different conformation than WT Non-denaturing PAGE, muts migrate different than WT How does EtBr cause DNA to fluoresce? Intercalates into the double helix Absorbs UV ~300 nm, emits ~600 nm Pulse Field Gel Electrophoresis steps 1. culture 2. embed pellet in agarose plug 3. treat w/ lysozyme (cell lysis) 4. proteinase K 5. gel What are the 3 steps of PCR and their temperatures? Denature 90-96C Anneal 50-70C Extension 68-75C Bisulfite DNA sequencing/Methylation specific 1. RE digest 2. Electrophorese and purify fragment of interest 3. Denature and incubate w/ sodium bisulfate (turns C>U, methylated C is unchanged) 4. clean, ppt, and resuspend 5. PCR --> sequence 6. Compare treated vs untreated, note where CG are not changed to TA NASBA steps 1. Hybridize oligo-T7P primer to target seq 2. RT/RNase H 3. Hybridize with target-specific oligo primer (P2) 4. RNA transcript of T7 RNA pol Do you have to know the gene sequence in order to do DNA sequencing? Yes, in order to design primers You do NOT need to know the mutation Sanger sequencing method 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 Fluorescent in situ hybridization (FISH) Uses fluorescent probes to detect DNA sequences on chr What types of probes are used for FISH? Dual fusion: 2 probes flank the breakpoint at both t locations CEN probes: centromeric probes bind to repetitive alpha satellite sequences Telomeric probes Whole chr paints What is the wavelength for background in spectrophotometery? 320 nm How do you determine quality of DNA/RNA using a spectrophotometer? A260/A280 What is considered good quality DNA/RNA from spectrophotometry? DNA: 1.7-2.0 RNA: 2.0-2.3 What is considered poor quality RNA/DNA from spectrophotometry? <1.7 Protein contamination What is one way you can increase the yield/quality of DNA/RNA after running gel? Do an EtOH ppt After an extraction/isolation, what should you elute with? DNA - TE or water RNA - DEPC water What are the two types of isolation/extraction methods? Liquid phase (organic & inorganic) Solid phase (Qiagen) Nucleotide Excision repair (NER) Done by endonucleases Removes a span of nt's by cleaving phosphodiester bond Base Excision repair (BER) Done by DNA glycosylase, AP endonuclease Cleaves glycosidic bond of a single base base, leaving apurinic/apyrimidinc site Describe the growth of the nucleic acid chain The chain grows by the attachment of the 5' phosphate group of an incoming nucleotide to the 3' hydroxyl group of the last nucleotide on the growing chain Denaturing agents formamide, urea, mercaptoethanol Solenoid a coil of six nucleosomes wound into a tightly packed helix Mutation DNA sequence change that is present in a relatively small proportion of the population <1%, somatic changes Variant inherited sequence alterations Polymorphism a change in the DNA sequence that is present in at least 1-2% of the population (ex. Sickle cell anemia) Gene mutations affect single genes and are often small changes in the DNA sequence Chromosome mutations Affects the structures of the entire chr, requires the movement of large chr regions Genome mutations Change in the number of chr's Eupliod normal complement of chromosomes Aneuploid Increased number of chr's (eg. Down's syndrome) Haploid Single copy of each chr (humans have 23) Diploid Two copies of each chr (humans have 46) DNA polymerase Catalyzes phosphodiester bond between nt's Uses ssDNA as a template to determine which nt's to add DNA Polymerase I (Prok) Processes Okazaki fragments Replaces RNA primers with DNA (exonuclease activity) Excision repair & proof reading DNA Polymerase II (Prok) DNA repair, exonuclease activity DNA Polymerase III (Prok) Primary enzyme involved in replication Exonuclease activity DNA Polymerase IV (Prok) Bypass replication SOS response DNA Polymerase V (Prok) Bypass replication SOS response Translesion synthesis DNA repair DNA Polymerase α (Euk) Primase DNA dependent DNA & RNA pol DNA Polymerase β (Euk) Base excision repair (BER) DNA Polymerase δ (Euk) Lagging strand synthesis DNA repair, exonuclease, replaces primers as it encounters Okazaki fragments DNA Polymerase ε (Euk) Leading strand synthesis exonuclease DNA Polymerase γ (Euk) mtDNA replication and repair Exonuclease activity Terminal transferase DNApol synthesizes poly-nt chain at 3' end w/o a template Transcription initiation --> elongation --> termination Retrotransposons Mobile genetic elements which can increase genome size and insert itself within coding/noncoding regions The three biochemical activities of reverse transcription RNA-dependent DNApol, Ribonuclease H, and DNA-dependent DNApol --> all used to create ds cDNA from RNA Describe the steps of reverse transctiption 1. tRNA acts as a primer and hybridizes to virus genome 2. Complementary DNA then binds to the U5 (non-coding region) and R region 3. RNAse H degrades the 5' end of the RNA which removes the U5 and R region. 4. The primer then "jumps" to the 3' end of the viral genome and the newly synthesized DNA strands hybrid Splicing modification of the nascent pre-messenger RNA (pre-mRNA) transcript in which introns are removed and exons are joined. R-factors resistance transfer factors. Carry antibiotic resistance to common antibiotics. Colicinogenic factors resistance to bacteriocins, toxic proteins manufactured by bacteria. Primary protein structure sequence of a chain of amino acids Secondary protein structure Amino acids are linked by H bonds to form sub structures; eg a helixes, B sheets Tertiary protein structure 3D structure of a single protein Quaternary protein structure 3D structure of a multi sub unit protein Restriction enzymes endonucleases that recognize specific sequences and break the phosphodiester bond of dsDNA Type I restriction enzymes have both nuclease and methylase activity in a single enzyme. Bind to host-specific DNA that contains methylated adenines Type III restriction enzymes resemble type I enzymes in their ability to methylate and restrict (cut) DNA. - adenine methylation occurs on only one strand. Type II restriction enzymes used most frequently in the laboratory - do not have inherent methylation activity in the same molecule as the nuclease activity Exonuclease I Degrades ssDNA from 3'-->5' Exonuclease III Removes 5' mono-nt's from the 3' end of the dsDNA in the presence of Mg2+ and Mn2+. Removes nucleotides from blunt ends, recessed ends, and nicks, but NOT overhangs! Exonuclease VII Degrades ssDNA from either the 5' or 3' ends One of the few enzymes with 5' exonuclease activity. Deoxyribonuclease I From bovine pancrease, digests ss and dsDNA at pyrimidines. Typically used to remove DNA from RNA preparations. Exonuclease II Proofreading function of the pol Degrades ssDNA from 3'-->5' Southern Blotting DNA is isolated and cut with REs. This allows investigators to determine the molecular weight of a restriction fragment and to measure relative amount in different samples. Southern Blotting Procedure 1. RE digest DNA 2. Gel electrophoresis 3. Soak in HCl (depurinates, weakens H-bonds) 4. Soak in NaOH (denatures) 5. DNA transferred to a membrane 6. Immobilize (UV or bake) 7. Pre hyb to block 8. Hyb with probe SyBr green Non-specific intercalation into the minor groove of dsDNA, can be used in qPCR Chaotropic agents disrupts the structure and denatures the DNA by increasing the entropy and non-covalent forces like hydrogen bonds (ex. chemicals like - sodium iodide, or sodium perchlorate) FRET probes Fluorescence Resonance energy transfer - Distance dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. Molecular beacons Measures accumulation of product at the annealing step Contains target specific seq and inverted repeat that forms stem-loop At annealing step probe hyb's to target, separating R and Q Scorpion probes PCR prod is covalently bound to dye; primer is bound to molc beacon type seq After extension, target specific seq will unfold w/ the newly synthesized target seq, separating R and Q Components of PCR DNA template Two primers that are complementary to the 3' Taq polymerase dNTPs Buffer solution Mg2+ (divalent cations) - higher Mn2+ concentration can increase the error rate during DNA synthesis Thermal Cycling steps in conventional PCR 1. Initialization (94-96 C) 2. Denaturation (94-98 C) 3. Annealing (50-65 C) 4. Extension (70-80 C) 5. Repeat 2-4 ~30x 6. Final Elongation (70-74 C) 6. Final hold Optimization of PCR methods 1. Check the Tm 2. Mg2+ concentration - too little can result in no PCR product, and too much may produce noise 3. PCR cycles 4. Add, extend, or increase the temp of the initial template denaturation step 5. Concentrations of other buffer components 6. GC Content 7. Taq concentration Real-Time PCR/qPCR PCR based method which is used to amplify and quantify a targeted DNA molecule. Enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when noramalized to the DNA input. PCR DNA Array DNA array is a collection of spots attached to a solid support (such as a microscope slide) where each spot contains one or more ssDNA oligo fragments. Arrays make it possible to put down large quantities of very small spots on a single slide. Each spot has a DNA fragment that is complementary to a single DNA sequence. Reverse Transcriptase PCR Used to detect RNA expression levels. RT-PCR is used to qualitatively detect gene expression through creation cDNA transcripts from RNA. Branched DNA Technololgy (bDNA) used for DNA or RNA; short probes are used to capture the target nucleic acid and then to multiple reporter molecules, loading the target nucleic acid with signal. Sequence Based Nucleic Acid Amplification (NASBA) Used to amplify RNA sequences; Primer-dependent technology that can be used for the continuous amplification of nucleic acids in a single mixture at one temperature; works at 41 C Transcription Mediated Technology (TMA) uses RNase H which degrades RNA from the intermediate hybride so denaturing is no longer required; Isothermal process - negates the requirement for thermal cycling to drive rxns. Targeting RNA allows for the direct detection of RNA viruses (HCV, HIV). Loop-mediated isothermal amplification (LAMP) Isothermal, single tube technique for the amplification of DNA Uses 4 different primers that recognize 6 regions on target gene Stem-loop structure forms which is used as a template for lamp cycling (SDA) Hybrid capture assays (HCA) Signal amplification Target DNA is released from the cell, denatured, and binds RNA probe DNA:RNA is recognized and binds Ab on solid support DNA:RNA are detected by adding Abs that bind w/ AP, substrate is added Cleavase/Invader Invader & signal probe added to target Cleavable substrate is formed if mutation is present Signal probe is cleaved to form invader in the next step FRET probe is added; if invader hybridizes, cleavase cuts flap, separating R and Q Sanger Sequencing Known as deoxy chain terminating sequencing; A primer complementary to the 5' region of DNA is used. The primer is typically labeled with P32 (internal labeling) or a fluorescent dye. Here, modified ddNTPs derivatives are added which lack the OH group found on the 3' carbon of the dNTPs. DNA synthesis will stop upon incorporation of a ddNTP because the bond between the phosphodiester bond cannot be established. 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. 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. 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. Dye terminator ddNTPs are used and fluorescently labeled instead of the primer. All four reactions are performed in the same tube. Terminated nucleotides are amplified. Dye primer Four different fluorescent dyes are added to the primers. Cycling is done to attach the primer and that is what the instrument sees. Each nucleotide is amplified a different color. Too many and too few ddNTPs result in: too many ddNTPS will result in many short sequence reads, too little will result in loss of sequencing data but will give a longer read. Maxam-Gilbert Sequencing Differing concentrations of salt are used in four different tubes - A, T, C, G - Usually DMS (dimethylsulphate), FA (formic acid), H (hydrazine), and H+S (hydrazine + salt) --> read on a gel Bisulfate DNA sequencing A type of chain termination sequencing designed to detect methylated nucleotides. Methylation of cytosine residues in DNA is an important part of gene regulation and expression - this is important for detecting different types of cancer. During the incubation C is converted to U and 5-methylated C is unchanged. A PCR reaction is then performed using normal chain termination methods. Denaturing High-performance Liquid Chromatography (HPLC) Analysis for PCR products 150-450 bp. The heteroduplexes elute ahead of the homoduplexes as the conditions intensify. The migrating homoduplexes are detected by absorbance at 260nm or fluorescence. Melt Curve Analysis 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. (FRET Probes - dissociation curves) Nucleic Acid labeling Common labels used to generate nucleic acid probes include radioactive phosphates, biotin, fluorophores and enzymes. In addition, the bioconjugation methods used for nucleic acid probe generation may be adapted for attaching nucleic acids to other molecules or surfaces to facilitate targeted delivery or immobilization, respectively. In-situ hybridization Used to detect protein, RNA, and DNA within the cell. Probes bind to the DNA and can be visualized under the microscope. Depending on the mutation, different signals can be seen - deletions and duplications. Sensitivity can be increased by using dual fusion probes, break apart probes, centromeric probes, and telomeric probes. Dual fusion probes Uses two pairs of probes with different fluor dye Bind regions that span the breakpoint of both t partners If t is present, signal from both dyes should be present Break apart probes Bind to the chr flanking the t breakpoint region WT will emit a combination signal (next to each other) and t will emit separated signals. Centromeric probes (CEN) designed to hybridize to the high alpha satellite sequences surrounding centromeres. Region specific to detect aneuploidy of chromosomes Telomeric probes Best for cryptic translocations or small abnormalities Single-strand conformation polymorphism (SSCP) Based on the preference of DNA to exist as DS rather than SS; Forms 3D conformers - a SNP can cause the conformer to fold differently (kinks, loops, bubbles, and tails) Restriction fragment length polymorphism (RFLP) Used to detect sequence alteration in retriction enzyme fragments; The region surrounding the mutation is amplified and the mutation is detected by cutting the amplicon with the correct restriction enzyme Analyte specific reagent (ASR) FDA defines analyte specific reagents (ASRs) in 21 CFR 864.4020 as "antibodies, both polyclonal and monoclonal, specific receptor proteins,ligands, nucleic acid sequences, and similar reagents which, through specific binding or chemical reaction with substances in a specimen, are intended to use in a diagnostic application for identification and quantification of an individual chemical substance or ligand in biological specimens. research use only (RUO) According to FDA, manufacturer-initiated studies of RUO products are typically intended to evaluate design, limited-scale performance, and issues such as usability of the test. The agency acknowledges that RUO products may be used for non-clinical laboratory research for goals other than developing a commercial IVD product. According to FDA, these uses may include developing novel and fundamental medical knowledge related to human disease and conditions. In-vitro diagnostics (IVD) Medical devices intended to perform diagnoses from assays in a test tube, or more generally in a controlled environment outside a living organism. lab developed tests (LDT) a term used to refer to a certain class of in vitro diagnostics (IVDs). In the United States, the Food and Drug Administration has determined that while such tests qualify as medical devices, FDA will allow these products to enter the market without prior approval from the Agency. CLIA Congress passed the Clinical Laboratory Improvement Amendments (CLIA) in 1988 establishing quality standards for all laboratory testing to ensure the accuracy, reliability and timeliness of patient test results regardless of where the test was performed. The Joint Commission An independent, not-for-profit organization, The Joint Commission accredits and certifies more than 20,000 health care organizations and programs in the United States. Joint Commission accreditation and certification is recognized nationwide as a symbol of quality that reflects an organization's commitment to meeting certain performance standards. CAP The College of American Pathologists (CAP), is a medical society serving more than 18,000 physician members and the global laboratory community. It is the world's largest association composed exclusively of board-certified pathologists and pathologists in training and is the worldwide leader in laboratory quality assurance. The College advocates accountable, highquality, and cost-effective patient care. CMS Previously known as the Health Care Financing Administration (HCFA), is a federal agency within the United States Department of Health and Human Services (DHHS) that administers the Medicare program and works in partnership with state governments to administer Medicaid, the State Children's Health Insurance Program (SCHIP), and health insurance portability standards. In addition to these programs, CMS has other responsibilities, including the administrative simplification standards from the Health Insurance Portability and Accountability Act of 1996 (HIPAA), quality standards in long-term care facilities (more commonly referred to as nursing homes) through its survey and certification process, and clinical laboratory quality standards under the Clinical Laboratory Improvement Amendments. CLSI Clinical Laboratory and Standards Institute; A not-for-profit membership organization, the Clinical and Laboratory Standards Institute (CLSI) brings together the global laboratory community for a common cause: fostering excellence in laboratory medicine. We do so by facilitating a unique process of developing clinical laboratory testing standards based on input from and consensus among industry, government, and health care professionals. FDA FDA is responsible for protecting the public health by assuring the safety, efficacy and security of human and veterinary drugs, biological products, medical devices, our nation's food supply, cosmetics, and products that emit radiation. also responsible for advancing the public health by helping to speed innovations that make medicines more effective, safer, and more affordable and by helping the public get the accurate, science-based information they need to use medicines and foods to maintain and improve their health. Methylation of cytosine bases 5' to the gene will increase or decrease expression? decrease Histone acetylation close to the gene will increase or decrease expression? increase siRNAs complementary to the gene transcript will increase or decrease expression? decrease Calculate the DNA concentration from the following: 260=0.172 (D.F. 1:100) 860 ug/mL = .172 abs 50 ug/mL 100 DF [DNA] = 1535 ug/mL. You have 0.5 mL. What is the total yield. 767.5 ug = 1535 ug/mL * 0.5mL [DNA] = 767.5 ug/mL. You have 0.5 mL What is the total yield. 383.75 ug = 767.5 ug/mL * 0.5 mL [DNA] = 860 ug/mL. You have 0.5 mL. What is the total yield. 430 ug = 860 ug/mL * 0.5mL Calculate the RNA concentration from the following: 260=0.307 (DF 1:100) 1228 ug/mL = 0.307 abs 40 ug/mL 100 DF An RNA preparation has the following readings: 260=0.208 280=0.096 Is this RNA suitable for use? Yes, 2.17 is suitable for RNA analysis A260/A280 = 0.208/0.096 How does PFGE separate larger fragments more efficiently than standard electrophoresis? Repeated reorientation forces larger fragments through the gel matrix more efficiently If fragments are dissolved in 50% formamide will the stringency be higher or lower? Higher If fragments are dissolved in a high concentration of NaCl will the stringency be higher or lower? Lower Does heating a solution from 65C to 75C during hybridization raise or lower stringency? raises [Show More]

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