Molecular Diagnostics Chapter 3 Study Questions with Verified Answers

Molecular Diagnostics Chapter 3 Study Questions with Verified Answers 1. Describes the general structure of an amino acid. - ANSWER Central carbon with carboxyl group, amino group, hydrogen, and a side chain 2. The chemical nature of alanine, leucine, phenylalanine, and tryptophan is described as what? - ANSWER Non polar 3. The enzyme that bonds two amino acids together in protein synthesis is what? - ANSWER Peptidyl transferase 4. What amino acid is used to initiate most proteins in eukaryotes? - ANSWER Methionine 5. A glycoprotein contains what attached after translation? - ANSWER Sugar moieties 6. Specialized proteins that bind to the large ribosomal subunit and protect the hydrophobic regions of growing polypeptides are called what? - ANSWER Molecular chaperones 7. In the ribosome, the growing peptide moves from site to site in what direction? - ANSWER A to P to E 8. Hydrolysis of the finished polypeptide from the final tRNA by release factors would occur at which of the following codons? - ANSWER UGA Which amino acid has a nonpolar R group? - ANSWER Valine 9. 260/280 ratio for pure DNA - ANSWER 1.6 to 2.0 times more, if greater than 2.0 could have RNA contamination, if less protein contamination. May need to refuter through column or repeat protein digestion, does not differentiate between heavily fragmented or degraded DNA will still get a reading 10.260/280 ratio for pure RNA - ANSWER 2.0-2.3 times more 11.Which dyes are used for DNA in fluorometry? Which are used for RNA? - ANSWER DNA: DABA, Hoechst 33258-combines with AT bp so specific for intact double-stranded DNA, Picogreen-most sensitive ds DNA detection, oligogreen- ssDNA detection RNA: sybergreen II (will also bind with DNA need to use DNase) 12.Compare and Contrast spectrophotometry and fluorometry - ANSWER Spectrophotometry: detects through absorption, does not require calibration with known sample, does not have time limit on read time, detects any nucleic acid even if degraded or nicked because will be absorbed by uv light, used for an array of procedures, RNA may be read through absorption and affect results ometry: detects through fluorescence of dyes, requires calibration with known sample, must be read within two hours, detects ds DNA that is intact, used for high sensitivity procedures like NGS, RNA will not be detected 14.What are two methods for separating nucleated cells? - ANSWER 1. density-gradient centrifugation of whole blood or bone marrow with ficoll 2. incubation of whole blood or bone marrow in hypotonic water which will lyse RBC's prior to WBC's, and then centrifuged to pellet the WBC's 15.Which fixative is the worst for DNA recover? - ANSWER Bouin's and B-5 16.Which fixative is the best for DNA recovery? - ANSWER buffered formalin 17.Steps of Organic DNA Extraction - ANSWER 1. lyse cells in suspension using alkaline (NaOH) and a detergent (SDS) 2. treat lysed debris with acetic acid and salt, centrifuge to pellet 3. add phenol and chloroform to separate solution into two layers an upper aqueous phase which contains the DNA and a lower organic phase which contains the cell debris ie proteins 4. precipitate DNA with ethanol, centrifuge, remove ethanol and reprecipitate with water/buffer, can add RNAse 18.What can be used to help precipitate low amounts of DNA? - ANSWER carrier molecules such as yeast, RNA, glycogen 19.How is quantity of DNA or RNA measured from gel electrophoresis? - ANSWER by using a control with a known quantity and comparing fluorescence intensity or densitometry 20.Why must RNA isolation use liquid nitrogen? - ANSWER to inactivate intracellular RNAses 21.What type ofRNA is typically required for analysis? How abundant is it? - ANSWER mRNA, 2.5-5% 22.How can specifically mRNA be isolated? - ANSWER by utilizing the polyA tail nature of mRNA. Columns with polyU oligos or polyT oligos attached will grad onto the poly A tail of mRNA. This helps to increase yield. 23.Fixed tissue and solid tissue are _________ for RNA recovery. Bone marrow and cultured cells are __________ for RNA recovery. - ANSWER bad, good 24.In early experiments, a polymer of uracil, UUUUUUUU..., translated into a peptide comprising only phenylalanine amino acids. What did this observation demonstrate? - ANSWER UUU codes for phenylalanine 25.The process of protein synthesis is also called what? - ANSWER Translation 26.The set of nucleotides in mRNA that encode one amino acid is called a(n) what? - ANSWER Codon 27.Which amino acid has a nonpolar R group? - ANSWER Valine 28.At physiological pH, amino acids are what? - ANSWER Zwitterions 29.Describes the general structure of an amino acid. - ANSWER Central carbon with carboxyl group, amino group, hydrogen, and a side chain