ASCP Molecular Biology Certification Exam(Technologist in Molecular Biology, MB(ASCP) board exam prep)

Pyrimidine One carbon ring Cytosine, Thymine, Uracil Purine Two carbon rings Adenine, Guanine 00:03 01:10 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