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65 Cards in this Set
- Front
- Back
How is DNA converted to RNA? |
Transcription |
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How is RNA converted to protein? |
Translation |
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What 5 methods are used to investigate DNA? |
Molecular Hybridisation: southern blotting, DNA sequencing, PCR, DNA fingerprinting, Microarrays |
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What 3 things does genomics discover? |
Identification of specific DNA signatures, prediction of restriction sites, DNA microarrays |
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What 5 methods investigate RNA? |
Molecular hybridisation: northern blotting, in situ analysis (FISH), RNA sequencing, microarrays, RT-PCR |
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What 4 things does transcriptomics investigate? |
Prediction of open reading frames, culture media design, prediction of virulence, gene expression in response to the environment |
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What 4 methods are used to investigate proteins? |
Molecular Hybridisation: western blotting, protein sequencing, mass spectrum, analysis |
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What 3 things are proteomics used for? |
Identification of antigenic epitopes, MAb production, vaccine design |
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What 4 things is genotyping useful for? |
quality control, description of laboratory cross-contamination, authentication of re-infection / relapse, investigation of infectious disease outbreak |
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What 3 approaches can be used for genotyping? |
pulsed-field gel electrophoresis, multi-locus sequence typing, whole genome mapping / sequencing |
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What does sub-typing discriminate? |
food-borne pathogens below species level |
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Why do sub-typing methods need to be reliable, sensitive and informative? |
match isolates with cases, discriminate between case isolates and unrelated isolates |
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Why is surveillance needed? 4 |
Identify new or emergent strains / clones, identify potential reservoirs of strains that cause disease, identify routes of transmission, improve knowledge of epidemiology of foodborne disease |
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What is the standardized approach to data acquisition and interpretation? |
PulseNet |
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What is bacterial sub-typing 1? |
identification to species or sub-species level, for some bacteria typing is part of identification |
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What is bacterial sub-typing 2? |
subtyping allows discrimination below species, subspecies |
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What is molecular sub-typing bacteria? |
characterization of DNA, RNA or protein of bacteria to discriminate below species, subspecies and types |
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HOw many subtypes of S. typhimurium are catalogued in PulseNet? |
over 1800
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Why is subtyping needed? 3 |
QC of fermented foods and pro/prebiotics, food processing applications, food-borne disease tracking |
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What are 3 sub-typing methods? |
Restriction fragment length polymorphism (RFLP) methods, PCR-based methods and DNA sequencing |
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What are the 4 types of RFLP based methods used to sub-type? |
Whole genome RFLP, ribotyping, pulsed field gel electrophoresis, whole genome mapping |
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What are the 3 PCR-based methods used for sub-typing? |
RAPD, REP and ERIC and multi-locus variable number of tandem repeats analysis (MLVA) |
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What are the 2 types of DNA sequencing used to sub-type? |
Multi-locus sequence typing (MLST) and whole genome sequencing |
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How can complexity of a DNA fingerprint be reduced? |
Pulsed-field gel electrophoresis (PFGE) |
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What does REA produce? |
large number of overlapping, poorly resolved DNA fragments |
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What is DNA macrorestriction analysis by PFGE? |
RFLP-based method that uses rare cutting restriction endonucleases to cut chromosomal DNA |
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Why are agarose plugs used in PFGE? |
DNA is digested in agarose plugs to prevent shearing |
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How are DNA fragments separated in PGFE? |
agarose gel |
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How is electricity involved in PGFE? |
Orientation of an electric field changes in a pulsed manner |
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What are macrorestricted profiles composed of? |
5-30 well resolved bands |
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What size are macrorestricted profiles? |
10-800kbp |
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What is the gold standard technique of sub-typing? |
PGFE |
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What restriction enzymes are used for E. coli? |
Xbal, Blnl, NotI |
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What are the 4 limitations of PGFE? |
specialized and expensive equipment, laborious and time consuming, not amenable to automation, interpretation of results can be difficult |
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What is likely to supersede conventional sub-typing? |
Whole genome sequencing |
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What happens during whole genome sequencing? |
Bacterial genome preparation is fragmented then sequenced using a suitable method |
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How is the genome assembled in WGS? |
Bioinformatic programs used, development of contigs first, then closure of remaining gaps |
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What is the genome used for? |
comparative studies to further characterize the bacterium, link genetic structure to phenotype |
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What are the 3 steps of traditional sequencing? |
Cloning, sanger sequencing-chain termination mechanism, electrophoresis with laser readout |
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What does NGS produce? |
shorter reads and more reads in shorter time |
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What does NGS sequence? |
individual clonally amplified DNA molecules |
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Does sanger or NGS have higher error rate? |
NGS |
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What does NGS provide? |
window to view real-time bacterial evolution |
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What is pyrosequencing? |
sequencing by synthesis methods, dNTP is incorporated and detected by photon emission |
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What does photon emission depend on? |
series of enzymatic reactions |
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How is ATP produced in pyrosequencing? |
PPi combines with adenosine phosphosulphate, catalysed by ATP sulphurylase |
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What produces light in pyrosequencing? |
luciferase, D-luciferin and O2 combine to make light |
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What does apyrase do in pyrosequencing? |
degrades any unincorporated dNTPs |
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What is important about E. coli's genome |
it is dynamic |
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What is S. agona's genome devoid of? |
plasmids |
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What type of antibiotics is E. coli O104 resistant to? |
b-lactam antibiotics |
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In what type of sells is there an epigenetic role of methylated bases? |
eukaryotic |
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What can methylate bases? |
bacteria as a function of their restriction modification systems |
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What do RM systems in bacteria do? |
Protect against deterious effects of partner restriction enzymes which act on transmissible DNA molecules, including phage |
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What are known to play a role in virulence? |
Methyl-transferases (MTases) |
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What detects methylated bases? |
SMRT sequencing |
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What is PFGE used for? |
tracking pathogens and identifying hot-spots of contamination |
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What can be identified by sub-typing in food industry? |
persistent clones that contaminate processing environment and the food production chain and may infect consumers |
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What 4 factors facilitate pathogen survival? |
growth rate in food matrices, heat resistance, resistance to biocides, virulence |
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What are the 9 characteristics of an idea sub-typing method? |
wide applicability, rapid results, high discrimination, reproducible, easy, automatable, inexpensive, ease of result interpretation, easy and automated comparison of patterns |
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What is the problem with genetic diversity? |
isolates differing by 1 DNA band may be very different |
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Can sub-typing prove or disprove a connection? |
multiple genotypes from food, co-infection |
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When are high clock speeds used? |
outbreak investigations |
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When are low clock speeds used? |
longitudinal studies |
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what is a standardized method? |
PFGE |