Download Molecular Biology: DNA, RNA, and Genetic Processes and more Exams Molecular biology in PDF only on Docsity!
ASCP Molecular Biology Certification
Exam
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
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]
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
- Isolate nucleic acid (RNA)
- Amplify
- Hybridization
- Strigent wash
- Incubate with conjugate
- Incubate with substrate
- 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
- isolate mRNA from cells
- RT to get labeled cDNA copies of mRNA
- cDNA washed over slide. cDNA sticks to comp sequence
- 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
- RE digest to fragment DNA
- Run on gel to separate
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
- culture
- embed pellet in agarose plug
- treat w/ lysozyme (cell lysis)
- proteinase K
- 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
- 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-
Bisulfite DNA sequencing/Methylation specific
- RE digest
- Electrophorese and purify fragment of interest
- Denature and incubate w/ sodium bisulfate (turns C>U, methylated C is unchanged)
- clean, ppt, and resuspend
- PCR --> sequence
- 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 Which pathogens can LCR be used to detect? Chlamydia Gonorrhea Listeria HPV Sickle cell dz Nucleic Acid Sequence Based Amplification (NASBA)
Isothermal, single tube rxn High sensitivity Enzymatic rxns take place concurrently NASBA steps
- 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? HIV Hepatitis HTLV CMV Transcription-Mediated Amplification (TMA) Two enzymes: RT and RNApol Isothermal RNA or DNA Strand Displacement Amplification (SDA) 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
ddNTP dideoxyribonucleoside triphosphate lack a hydroxyl group (OH) at 2' and 3' Fluorescent in situ hybridization (FISH) Uses fluorescent probes to detect DNA sequences on chr What are some of the benifits of FISH? Large number of cells may be scored Dual color --> multiple targets Many sample types What types of probes are used for FISH? 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? 320 nm How do you determine quality of DNA/RNA using a spectrophotometer? A260/A What is considered good quality DNA/RNA from spectrophotometry? DNA: 1.7-2. RNA: 2.0-2. What is considered poor quality RNA/DNA from spectrophotometry? <1.7 Protein contamination
What does smearing on a gel indicate? Sample degradation Loaded too much How can you tell if you have good RNA using gel electrophoresis? 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? Do an EtOH ppt Which specimen tubes are the best for use with molecular assay? EDTA (lavender/purple) ACD (yellow) Which specimen tubes are not good for use with molecular assays? 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? NO Room temp 22-25C Neutrophils will degranulate if frozen What is the best temperature to store DNA? Long term = -70C short term = -20C For long term storage, what should you store DNA in? TE or DNase free H2O
Diagnostic sensitivity likelihood of positives Diagnostic specificity likelihood of negatives Clinical sensitivity and specificity is based on _________. outcome Analytical sensitivity is based on _____ and specificity is based on _______. limit of detection target specificity What is direct analysis? identifying a specific gene or mutation that caues disease. gene must be known, might need to know mut What are some direct analysis methods? southern PCR - ASO blot, restriction digest SSCP HA What some indirect analysis methods? RFLP linkage analysis What is indirect analysis? inherited marker near gene associated with disease unknown gene and /or mut
Describe the growth of the nucleic acid chain 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 formamide, urea, mercaptoethanol Histones Proteins that DNA tightly coils around to form chromosomes Octamer, 2 ea of: H2A, H2B, H3, and H Histones are hydrophobic (basic K & R,+) DNA is hydrophilic (P backbone, -) +/- interaction keeps them bound Solenoid a coil of six nucleosomes wound into a tightly packed helix Mutation DNA sequence change that is present in a relatively small proportion of the population <1%, somatic changes Variant inherited sequence alterations Polymorphism a change in the DNA sequence that is present in at least 1-2% of the population (ex. Sickle cell anemia) Gene mutations affect single genes and are often small changes in the DNA sequence
