ASCP Molecular Biology Certification Exam Questions and Answers 100% correct |2023

ASCP Molecular Biology Certification Exam Questions and Answers 100% correct |2023 Pyrimidine One carbon ring Cytosine, Thymine, Uracil Purine Two carbon rings Adenine, Guanine How are nucleotides joined together? Condensation to form phosphodiester bond 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 Exonucleases Degrades nucleic acids by removing one terminal nt at a time Cleaves phosphodiester bond at end of chain 5' --> 3' and 3' --> 5' 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 Primase DNApol α (DNA dep RNA pol) adds short segments of complementary RNA to ssDNA template (primers), serves as starting points for replication single-strand DNA binding proteins (SSBPs) Binds ssDNA and prevents it from re-annealing during TXN, replication, repair, and recombination Okazaki fragments Short fragments of DNA synthesized by DNApol δ using the lagging strand (3'->5') as a template Ligase Closes gaps in DNA Catalyzes phosphodiester bond between 3'OH and 5'P What are the steps in DNA replication? 1. Initiate 2. Elongate 3. Terminate 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 Poly-A tail Prevents mRNA from being degraded in cytoplasm 100-250 A's at 3' end 5' cap 5'-5' pyrophosphate bridge to a methylated G added to 5' end of a mRNA Protects against degradation and as a recognition signal for TLN apparatus aminoacyl tRNA tRNAs that carry amino acids Ribosomes Where TLN occurs Prok: 30s and 50s Euk: 40s and 60s Catalyzes peptide bond between a.a.'s What is the path of a tRNA in a ribosome? Acceptor > Peptidyl > Exit How is translation initiated? small rRNA (40S) subunit binds mRNA and scans for start codon (AUG) Met-tRNA is brought to the P site Large rRNA (60S) subunit binds How is translation terminated? Occurs when stop codon enters A site Release factor recognizes stop codon, hydrolyzes ester bond with P site, releasing aa chain Reverse transcriptase enzyme that transcribes RNA to cDNA (lacks introns) RNA --> RNA:DNA --> cDNA (dsDNA) Pleiotrophy a single gene controls the expression of many phenotypic traits ie Sickle Cell Anemia cDNA intron free complementary DNA can be inserted into a plasmid Vector helps carry DNA into cell ie plasmids, virus 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 How do you inactivate RNases? 200C for 2 hrs 30 min in 1M NaOH or quanidinum isothiocyanate Hybridization 2 ssDNA molecules of comp base sequence can form a ds hybrid (duplex) What does the incubation step in hybridization do? Allows formation of ds molecules Blocking DNA (Hybridization) minimizes probe binding to nonspecific sequence ie salmon sperm DNA, Human LINE-1 Blocking Proteins (Hybridization) minimize nonspecific binding of probe to membrane ie casein (milk), Denhardt's sol Stringency conditions of hybridization that control the specificity of binding of the probe to the target sequence How can you increase strigency in a hybridization? decrease [salt] increase [formamide] increase temp 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 Line Probe Assay steps 1. Isolate nucleic acid (RNA) 2. Amplify 3. Hybridization 4. Strigent wash 5. Incubate with conjugate 6. Incubate with substrate 7. Detect What method would you use if you knew the gene sequence and the mutation? Reverse Dot Blot Microarrays Used for unknown gene and mutation cDNA libraries can be used for gene expression, tumors, genetic mapping, mutations and polymorphism large scale, high throughput analysis Microarray steps 1. isolate mRNA from cells 2. RT to get labeled cDNA copies of mRNA 3. cDNA washed over slide. cDNA sticks to comp sequence 4. use laser to read fluorescent tags Southern Blot Detect a large DNA fragment among many Target: DNA, probe: DNA What can Southern Blots be used to detect? Deletions/insertions Point mutations Polymorphisms Structural rearrangements Southern Blot steps 1. RE digest to fragment DNA 2. Run on gel to separate 3. Soak gel in alkali/NaOH to denature dsDNA 4. Transfer ssDNA fragments to positively charged membrane (blot) 5. Fix to filter by heat (80C) or UV crosslink 6. Incubate (hybridize) blot w/ radioactively labeled ssDNA comp probe 7. Autoradiograph Heterduplex Analysis (HA) use for known gene, unknown mutation mutation screening bands on gel --> retarded migration from WT due to seq differences Heterduplex Analysis steps 1. PCR 2. Mix sample and CTR DNA together 3. Denature PCR using heat 4. Cool slowly to rt 5. Add denaturing loading buffer 6. Run on MDE gel 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 What makes DNA negatively charged? Phosphate groups of the phosphate:ribose backbone How does EtBr cause DNA to fluoresce? Intercalates into the double helix Absorbs UV ~300 nm, emits ~600 nm TAE Buffer tris-acetate w/ EDTA good for DNA recovery good for lrg fragments low buffering capacity increases migration of DNA thru gel TBE Buffer tris-borate w/ EDTA good for small DNA fragments high buffering capacity decreases migration thru gel Pulse Field Gel Electrophoresis steps 1. culture 2. embed pellet in agarose plug 3. treat w/ lysozyme (cell lysis) 4. proteinase K 5. gel How can Tween 20 affect PCR? Stabilizes Taq Suppress formation of 2* structures Increase yield Increase non-specific amplification What are some disadvantages of PCR? Must know the sequence first Prone to contamination May not be 100% specific Specificity dependent on temp and [Mg] What is the purpose of primers in PCR? to intiate replication What are the 3 steps of PCR and their temperatures? Denature 90-96C Anneal 50-70C Extension 68-75C PCR process 1. Prep MMx: buffer, taq, primers, dNTPs 2. Add target 3. Place in thermocycler 4. Denature dsDNA 5. Anneal: allows primers to hyb 6. Extend: pol adds dNTPs to 3' 7. Repeat steps 4-6 What can cause primer dimers? annealing temp too low too much primer What are primer dimers? Size is sum of two primers Primers hyb and are extended by Taq What can inhibit PCR amplification? Detergent (SDS) Phenol (left over from DNA isolation) Heparin (specimen tube) Heme Dyes CSF, urine, sputum, parafilm What is the use of uracil-N-glycosylase (UNG) in PCR? Prevents contamination by destroying amplicons containing dUTPs What are some causes of too many bands on a gel after PCR? Primers not specific Annealing temp too low Too many cycles Too much Mg++ RT-PCR RNA --> cDNA first strand synthesis 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 How does PCR work with methylated DNA? primers are designed to recognize methylated and unmethylated sense strands at gene promoter the methylated bases inhibit enzyme activity at recognition sites Ligase Chain Reaction (LCR) Probe amplification Two adjacent primers are ligated and amplified by a 2nd set of primers if mutation is present at 3' end of upstream primer Can detect a 1 bp mis-match