Molecular Epidemiology Flashcards
What is molecular epidemiology?
Application of techniques of molecular biology to epidemiology (i.e. study of distribution/determinants of disease, and the application of this study to control of diseases and other health problems)
The four aspects of molecular epidemiology.
Identification and diagnosis, resistance/susceptibility, phylogenetic relationships, population structure.
Identification and diagnosis
Detection and characterisation of infections
Monitoring parasites in the environment
Resistance/susceptibility
Molecular basis of resistance
Mapping spread of resistance alleles
Phylogenetic relationship
Origin and dispersal of pathogens
Discrimination between species/strains
Population structure
Levels of gene flow between populations
Predicting spread of heritable traits
Why target organelle DNA/amino acid sequences?
High copy number within a cell
Many complete genomes known
Design of universal primers
Why target nuclear DNA sequences?
Highly repetitive elements or single copy genes
DNA sequences have different evolutionary dynamics
What is the purpose of real-time PCR?
An assay that monitors accumulation of a DNA product from a PCR reaction in real time. Can quantify the amount of DNA in a sample at the start of a reaction. Increasing fluorescence as band amplified in real time.
Real-time PCR process
An oligonucleotide probe is added to the reaction (fluorescent reporter and quencher, the quencher reduced fluorescence intensity)
During PCR the probe denatures and anneals to the target sequence.
For every amplification, a fluorescence probe is released following complete polymerisation of DNA strand by Taq
Three primary steps of PCR
Denaturing, annealing and elongating
What is LAMP
Loop-mediated isothermal amplification. A target sequence is amplified with 2/3 sets of primers and a polymerase with high strand displacement activity (as well as replication activity)
Advantages of LAMP over PCR
Constant temperature. No thermal cycler. Higher amount of DNA produced (nature of loop primers). Can conduct in field.
How is DNA detected in LAMP?
Turbidity - (by photometry) by increased levels of magnesium pyrophosphate precipitate
Intercalation or direct labelling of molecule - DNA-binding dyes (SYBR green (naked eye)/SYTO 9 (fluorescence))
What are isoenzymes?
Enzymes which differ in their amino acid sequence but catalyse the same reaction.
How are isoenzymes analysed?
Protein from the DNA separated by electrophoresis. Use dyes which respond to enzyme activity to identify. Electrophoresis will run at different time lengths depending on the species.
AFLP process.
Amplified fragment length polymorphism.
Genomic DNA extract is digested with a restriction enzyme (EcoR1, BamH1). Adapters are ligated to the end of restriction fragments and PCR amplifies using primers complimentary to the adaptor. Bands are visualised either on a agarose or capillary gel electrophoresis.
What does AFLP show?
Genetic variation across a whole genome
Microsatellite analysis process.
The genomic DNA extract is run through PCR to amplify the microsatellite loci using fluorescently labelled 5’ primer. Capillary electrophoresis is used to determine fragment sizes NOT GELS
What does microsatellite analysis show?
Repetitive regions in genome that can vary In size. These regions are non-coding.
What does SNP analyse?
Single nucleotide polymorphisms look for one base change. It may be associated with drug resistance.
Expensive detection techniques
PCR and sequencing, NGS sequencing
Moderately priced techniques
Real-time PCR, SNP microarray
Cheap detection techniques
RFLP and PCR-RFLP
