ASCP Molecular Biology Certification Exam Technologist in Molecular Biology MB ASCP Board Exam Prep 2024 Pyrimidine - Answer One carbon ring Cytosine, Thymine, Uracil What is the function of mRNA? - Answer Carries genetic info out of nucleus Transcript translated to protein What is the function of tRNA? - Answer Carries aa to ribosome Anticodon pairs with codon on mRNA strand What is the function of rRNA? - Answer part of ribosome structure most abundant RNA coordinated coupling of tRNA to mRNA codons Feedback inhibition - Answer Product of pathway is noncompetitive inhibitor Binds to allosteric site to slow down rxn b/c too much product Endonucleases (Prok) - Answer Restriction enzymes Cleaves phoshpodiester bonds w/i poly -nt chain Recognition site is palindromic sequence Types I -V ORI sites - Answer nt sequence where replication is initiated Topoisomerase I - Answer 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 - Answer cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then reanneals the cut strands Gyrase (topoisomerase II) - Answer Unwinds supercoiling caused by unwinding at the rep fork by introducing DSBs Helicase - Answer Breaks H -bonds of double helix at the replication fork single -strand DNA binding proteins (SSBPs) - Answer Binds ssDNA and prevents it from re -annealing during TXN, replication, repair, and recombination Telomeres - Answer Repeat sequence (TTAGGG) at the ends of chr, protect chr from degradation RNA polymerase - Answer DNA dependent RNApol Transcribes DNA template to RNA (3' -->5'; anti -parallel) Splicesomes - Answer Complex of snRNPs Removes introns from pre -mRNA and splices exons together Enhancers - Answer Short regions of DNA that bind proteins (TXN factors) that enhance TXN of a gene Reverse transcriptase - Answer enzyme that transcribes RNA to cDNA (lacks introns) RNA --> RNA:DNA --> cDNA (dsDNA) Open Reading Frame (ORF) - Answer 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 - Answer Measures amount of light absorbed Quantitative measurement of [DNA/RNA] At what wavelength does DNA and RNA absorb? - Answer 260 nm At what wavelength does protein absorb? - Answer 280 nm 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 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? - Answer Allows formation of ds molecules Formamide acts as a __________ in a hybridization. - Answer denaturing agent Line Probe Assay (LiPA) - Answer reverse hybridization assay using sequence -
specific oligonucleotide probes (reverse SSOP) multi -parameter testing --> single strip Single -Stranded Conformational Polymorphism Ananlysis (SSCP) - Answer 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? - Answer Intercalates into the double helix Absorbs UV ~300 nm, emits ~600 nm Pulse Field Gel Electrophoresis steps - Answer 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? - Answer Denature 90 -96C Anneal 50 -70C Extension 68 -75C Bisulfite DNA sequencing/Methylation specific - Answer 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 - Answer 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? - Answer Yes, in order to design primers You do NOT need to know the mutation Sanger sequencing method - 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 Fluorescent in situ hybridization (FISH) - Answer Uses fluorescent probes to detect DNA sequences on chr What types of probes are used for FISH? - Answer 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? - Answer 320 nm How do you determine quality of DNA/RNA using a spectrophotometer? - Answer A260/A280 What is considered good quality DNA/RNA from spectrophotometry? - Answer DNA: 1.7-2.0 RNA: 2.0 -2.3 What is considered poor quality RNA/DNA from spectrophotometry? - Answer <1.7 Protein contamination What is one way you can increase the yield/quality of DNA/RNA after running gel? - Answer Do an EtOH ppt After an extraction/isolation, what should you elute with? - Answer DNA - TE or water RNA - DEPC water What are the two types of isolation/extraction methods? - Answer Liquid phase (organic & inorganic) Solid phase (Qiagen) Nucleotide Excision repair (NER) - Answer Done by endonucleases Removes a span of nt's by cleaving phosphodiester bond Base Excision repair (BER) - Answer 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 - Answer 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 - Answer formamide, urea, mercaptoethanol Solenoid - Answer a coil of six nucleosomes wound into a tightly packed helix Mutation - Answer DNA sequence change that is present in a relatively small proportion of the population <1%, somatic changes Variant - Answer inherited sequence alterations Polymorphism - Answer a change in the DNA sequence that is present in at least 1 -
2% of the population (ex. Sickle cell anemia) Gene mutations - Answer affect single genes and are often small changes in the DNA sequence Chromosome mutations - Answer Affects the structures of the entire chr, requires the movement of large chr regions Genome mutations - Answer Change in the number of chr's Eupliod - Answer normal complement of chromosomes Aneuploid - Answer Increased number of chr's (eg. Down's syndrome)
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