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ASCP Molecular Biology Certification
Exam
Pyrimidine - ANS>One carbon ring Cytosine, Thymine, Uracil Purine - ANS>Two carbon rings Adenine, Guanine How are nucleotides joined together? - ANS>Condensation to form phosphodiester bond What is the function of mRNA? - ANS>Carries genetic info out of nucleus Transcript translated to protein What is the function of tRNA? - ANS>Carries aa to ribosome Anticodon pairs with codon on mRNA strand What is the function of rRNA? - ANS>part of ribosome structure most abundant RNA coordinated coupling of tRNA to mRNA codons
Feedback inhibition - ANS>Product of pathway is noncompetitive inhibitor Binds to allosteric site to slow down rxn b/c too much product Exonucleases - ANS>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) - ANS>Restriction enzymes Cleaves phoshpodiester bonds w/i poly-nt chain Recognition site is palindromic sequence Types I-V ORI sites - ANS>nt sequence where replication is initiated Topoisomerase I - ANS>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
What are the steps in DNA replication? - ANS>1. Initiate
- Elongate
- Terminate Telomeres - ANS>Repeat sequence (TTAGGG) at the ends of chr, protect chr from degradation RNA polymerase - ANS>DNA dependent RNApol Transcribes DNA template to RNA (3'-->5'; anti-parallel) Splicesomes - ANS>Complex of snRNPs Removes introns from pre-mRNA and splices exons together Enhancers - ANS>Short regions of DNA that bind proteins (TXN factors) that enhance TXN of a gene Poly-A tail - ANS>Prevents mRNA from being degraded in cytoplasm 100-250 A's at 3' end 5' cap - ANS>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 - ANS>tRNAs that carry amino acids Ribosomes - ANS>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? - ANS>Acceptor > Peptidyl > Exit How is translation initiated? - ANS>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? - ANS>Occurs when stop codon enters A site Release factor recognizes stop codon, hydrolyzes ester bond with P site, releasing aa chain
Quantitative measurement of [DNA/RNA] At what wavelength does DNA and RNA absorb? - ANS>260 nm At what wavelength does protein absorb? - ANS>280 nm Organic isolation method - ANS>1. Lyse
- Add phenol/ chloroform > vortex/spin
- Transfer aqueous layer (top) to new tube
- Add chloroform:IAA (removes phenol) > vortex/spin
- Transfer aqueous layer to new tube
- Add NaOAc and EtOH > vortex/spin
- Decant
- Resuspend How do you inactivate RNases? - ANS>200C for 2 hrs 30 min in 1M NaOH or quanidinum isothiocyanate Hybridization - ANS>2 ssDNA molecules of comp base sequence can form a ds hybrid (duplex)
What does the incubation step in hybridization do? - ANS>Allows formation of ds molecules Blocking DNA (Hybridization) - ANS>minimizes probe binding to nonspecific sequence ie salmon sperm DNA, Human LINE- Blocking Proteins (Hybridization) - ANS>minimize nonspecific binding of probe to membrane ie casein (milk), Denhardt's sol Stringency - ANS>conditions of hybridization that control the specificity of binding of the probe to the target sequence How can you increase strigency in a hybridization? - ANS>decrease [salt] increase [formamide] increase temp Formamide acts as a __________ in a hybridization. - ANS>denaturing agent Line Probe Assay (LiPA) - ANS>reverse hybridization assay using sequence-specific oligonucleotide probes (reverse SSOP)
- use laser to read fluorescent tags Southern Blot - ANS>Detect a large DNA fragment among many Target: DNA, probe: DNA What can Southern Blots be used to detect? - ANS>Deletions/insertions Point mutations Polymorphisms Structural rearrangements Southern Blot steps - ANS>1. RE digest to fragment DNA
- Run on gel to separate
- Soak gel in alkali/NaOH to denature dsDNA
- Transfer ssDNA fragments to positively charged membrane (blot)
- Fix to filter by heat (80C) or UV crosslink
- Incubate (hybridize) blot w/ radioactively labeled ssDNA comp probe
- Autoradiograph Heterduplex Analysis (HA) - ANS>use for known gene, unknown mutation mutation screening
bands on gel --> retarded migration from WT due to seq differences Heterduplex Analysis steps - ANS>1. PCR
- Mix sample and CTR DNA together
- Denature PCR using heat
- Cool slowly to rt
- Add denaturing loading buffer
- Run on MDE gel Single-Stranded Conformational Polymorphism Ananlysis (SSCP) - ANS>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? - ANS>Phosphate groups of the phosphate:ribose backbone How does EtBr cause DNA to fluoresce? - ANS>Intercalates into the double helix Absorbs UV ~300 nm, emits ~600 nm
Increase non-specific amplification What are some disadvantages of PCR? - ANS>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? - ANS>to intiate replication What are the 3 steps of PCR and their temperatures? - ANS>Denature 90-96C Anneal 50-70C Extension 68-75C PCR process - ANS>1. Prep MMx: buffer, taq, primers, dNTPs
- Add target
- Place in thermocycler
- Denature dsDNA
- Anneal: allows primers to hyb
- Extend: pol adds dNTPs to 3'
- Repeat steps 4-
What can cause primer dimers? - ANS>annealing temp too low too much primer What are primer dimers? - ANS>Size is sum of two primers Primers hyb and are extended by Taq What can inhibit PCR amplification? - ANS>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? - ANS>Prevents contamination by destroying amplicons containing dUTPs What are some causes of too many bands on a gel after PCR? - ANS>Primers not specific Annealing temp too low Too many cycles Too much Mg++
Which pathogens can LCR be used to detect? - ANS>Chlamydia Gonorrhea Listeria HPV Sickle cell dz Nucleic Acid Sequence Based Amplification (NASBA) - ANS>Isothermal, single tube rxn High sensitivity Enzymatic rxns take place concurrently NASBA steps - ANS>1. Hybridize oligo-T7P primer to target seq
- RT/RNase H
- Hybridize with target-specific oligo primer (P2)
- RNA transcript of T7 RNA pol What pathogens can you detect using NASBA? - ANS>HIV Hepatitis HTLV CMV
Transcription-Mediated Amplification (TMA) - ANS>Two enzymes: RT and RNApol Isothermal RNA or DNA Strand Displacement Amplification (SDA) - ANS>1st stage: target generated w/ RE site 2nd stage: probe amp; HincII nicks at RE site, DNApol extends/regenerates RE site and displaces strand high throughput high sensitivity Branched DNA (bDNA) - ANS>Signal amplification Target captured by probes on a solid support Extender, pre-amp, and amp probes hybridized Amp bind alkaline phosphatase Dioxetane is added as a substrate for chemiluminescence Measured with a luminometer What are the clinical uses for DNA sequencing? - ANS>Mutation detection Confirm mutation by other method
What are some of the benifits of FISH? - ANS>Large number of cells may be scored Dual color --> multiple targets Many sample types What types of probes are used for FISH? - ANS>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? - ANS>320 nm How do you determine quality of DNA/RNA using a spectrophotometer? - ANS>A260/A What is considered good quality DNA/RNA from spectrophotometry? - ANS>DNA: 1.7-2. RNA: 2.0-2.
What is considered poor quality RNA/DNA from spectrophotometry? - ANS><1.7 Protein contamination What does smearing on a gel indicate? - ANS>Sample degradation Loaded too much How can you tell if you have good RNA using gel electrophoresis? - ANS>Good RNA will have a 2:1 intensity (28S : 18S) if 18S is more, RNA degradation is possible What is one way you can increase the yield/quality of DNA/RNA after running gel? - ANS>Do an EtOH ppt Which specimen tubes are the best for use with molecular assay? - ANS>EDTA (lavender/purple) ACD (yellow) Which specimen tubes are not good for use with molecular assays? - ANS>Heparin (brown/green) inhibits several enzymes used in molecular assays Should you freeze blood or bone marrow if you are going to use it in a molecular assay? - ANS>NO
