PhD week 33: Going long

Diagram showing the arrangement of genes in the mitochondrial genome of Drosophila yakuba. This is the typical arrangement for insects.

Mitochondria are organelles in the cell that provide energy-carrying molecules that are used in other cell functions. They do this in a manner somewhat analogous to a hydroelectric power station by creating an electrochemical gradient across a membrane, then using this gradient to power a turbine that produces the molecules. It's a place where a lot of activity is happening, and it needs a repair crew immediately on hand to keep it running. For this reason, there is a small amount of DNA inside the mitochondrion that contains the vital proteins and RNA units that are required for optimal performance.

Because there are a lot of mitochondria in each cell, mitochondrial DNA tends to be easily amplified from a range of organisms, making mitochondrial genes the regions of choice for phylogenetic analyses. One gene in particular, cytochrome c oxidase subunit 1 (COI), has been earmarked as being the gene of choice for specimen identification or DNA barcoding as it is otherwise known. For both these reasons, I am keen to use this gene in my analyses of Irenimus relationships. However, I'm finding that this is more easily said than done. It seems that entimine weevils have a lot of variation in these genes, which makes them hard to amplify. One idea that I will be trying involves trying to amplify the 5000 base pair region between the 12S gene (which is often less variable between species) and COI. A schematic is shown above.

To assist in planning this endeavour, I am able to make use of the resources available on Genbank. Genbank is a repository for DNA sequence data collected by scientists around the world. One can use this data to get an idea of where might be good places to position PCR primers, which provide the starting points for amplification. Unfortunately, there are only two complete weevil mitochondrial genomes available on Genbank—For Naupactus xanthographus and an unidentified Sphenophorus species. In addition, there are partial genomes available for 26 other species. However, these partial genomes cover everything but the region that I'm wanting to use, so are rather uninformative for my purposes. It looks like my plan going forward is just going to have to involve going in blind.

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Read:
   Mazur MA. 2012. First record of the tribe Eugnomini Lacordaire 1863 (Coleoptera: Curculionidae) from Fiji, with description of Pactola fiji sp. n.Zootaxa 3517: 63–70
   Psalms 130–131

Websites:
Renaming files in Linux
Inkscape Manual—Patterns along paths
Wikipedia—The Planets
Half of all wetlands destroyed since 1900