Epidemiology/Evolution Flashcards
What is RFLP PCR? Give an example of its use, advantages and disadvantages
Restriction Fragment Length Polymorphism PCR creates multiple copies of certain restriction fragments of DNA ( cut with restriction enzymes such as Hind 3) which can be compared for differences between isolates. One such example is Doudi et al. (2010) who use PCR RFLP to compare Leishmania major & L. tropica isolates obtained in Isfahan and Bam, Iran. The Internal Transcribed Spacer 1 region was amplified, and two isolates for each species discovered. Group A for L. major was common in Isfahan, with L. tropica group A being most prevalent in Bam
What are the 4 steps of general PCR?
- Denaturation - heating of components (template DNA, primers, nucleotides, mono/divalent cations, buffer solution, DNA polymerase) to 94-98°C for 20-30s to disrupt H binds, making DNA single stranded
- Annealing - temperature decreases to 50-65°C for 20-40s to allow for annealing of primers to template
- Elongation - temperature increases to 70-75°C allowing DNA polymerase to synthesise new complementary strands on template by addition of nucleotides
- Repeat - cycles are repeated 20-40 times to use all reagents and amplify DNA
What is RAPD? Give an example of use, it’s advantages and disadvantages
Random Amplification of Polymorphic DNA. Can be used as a multi-locus approach that only requires a small amount of DNA to assess relatedness of stocks. A short, single primers randomly binds to the DNA sample and PCR commences to amplify this template. Bands are created for the stocks that can be compared and matched to relatedness. Not possible to interpret genetically or mixed source, and is easily contaminated. Used by Jamonneau et al. (2002) to show genetic homology within a type 1 foci of Trypanosoma brucei gambiense, and that they are distinguishable from T. b. brucei and T. b. rhodesiense
What is MGE-PCR? Give an example of its use and its advantages/disadvantages
Mobile Genetic Element PCR was developed to over come problems encountered with RAPD/AFLP. Utilises the positional variation of MGEs to detect genetic variation. A primer designed for the RIME MGE of T. brucei allows for amplification of bands between RIME elements, producing banding patterns that can be compared. Differences can be combined with numerical taxonomy to identify stocks and was used by Tilley et al. (2003) when looking at samples from southeast Uganda, Zambia & Kenya for infective and non-infective stocks. Advantages include; PCR-based, specific for T. brucei DNA, multi locus in analysis, represents a significant portion of the genome and generates a single footprint. Disadvantages; cannot detect mixed samples and be interpreted genetically
How are micro-/mini- satellite markers used? Give an example and list advantages/disadvantages
Mini and micro satellites are short repeated sequences that vary in length or sequence between trypanosome. These can be amplified and separated by gel electrophoresis for strain comparison. Advantages; detect mixed infections, don’t require amplification in rodents and are highly specific/sensitive for T. brucei. Used by MacLeod et al. (2001) to study differences between geographical populations in Uganda & Zambia and Jamonneau et al. (2004) to discover a new type of T. b. gambiense associate with a symptomatic disease in humans
What are the three steps that are conducted to study evolution on a molecular basis?
DNA sequencing can be used to compare evolution between species. Look at the same gene and compare.
- Align sequences of the species
- Draw a similarity matrix that expresses as a percent the amount of bases that are the same
- Convert these percentages to a phylogenetic tree (can be rooted, unroofed or a cladogram). Similarity along the bottom then branch off. Rooted trees can be used to compare other species as branches can be added, e.g. when looking at human evolution, can add dogs to see relatedness. Unrooted trees are used when organisms aren’t known and measure difference i.e. the longer the branch, the greater the difference
What genes are used for evolutionary comparison?
Genes compared need homology in function. The most useful are protein synthesis genes. Ribosomal RNA genes are highly conserved in function but can differ in sequence. They are repeated but number varies e.g humans several hundred, frogs have thousands. Within the repeats are two units (ribosome made of 2 subunits) gene for each subunit. Large gene -> internal transcribed spacer -> 5.8s RNA -> ITS -> small gene. Spacer regions (gaps between gene repeats) are compared within species and are non conserved. ITS are flanked by the conserved genes, but the ITS aren’t conserved so can vary between strains
Give an example of a host-parasite evolution and the reason why its useful
Trypanosoma brucei brucei - animals
T. b. gambiense - humans in W. Africa
T. b. rhodesiense - humans in E. and central Africa
T. b. species can’t live outside tsetse fly
T. evansi is closely related to T. b. - infects camels and is transmitted mechanically by insects i.e. doesn’t need to be taken up by insect and change in midgut. They stay on proboscis and transmit directly so can bypass tsetse fly which increases its geographical range. Loss of DNA in kinetoplast caused this change, now exists in Africa, Asia, S. America
T. equiperdum is another that has evolved to by pass all insects and is transmitted sexually. Found in Africa, S. America, Asia
This is important as it shows how species can evolve transmission routes, increasing location. Single mutation of SRA gene caused T. b. brucei to become human infective (T. b. rhodesiense). Potential of host transmission and then infectivity.
PFGE as a tool for molecular epidemiology
Pulse-field Gel Electrophoresis allows for millions of bp sequences to be compared. Over a series of days, chromosomes are extracted from organisms set in a gel. The resulting contents are placed in a well and electrophoresis is conducted. Instead of a single direction, this time the voltage is direction on a central axis, and two running 60° either side. Pulse times are the same, but as longer DNA moves slower, the change in direction separated varying lengths, overcoming the normal 30-50kb limit of normal electrophoresis
