Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...
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Identification <strong>of</strong> putative odorant receptors from Epiphyas postvittana 80<br />
the cod<strong>in</strong>g RNA and prote<strong>in</strong> (Sterky and Lundeberg, 2000) and the references with<strong>in</strong>).<br />
Therefore, sequenc<strong>in</strong>g <strong>of</strong> cDNA, which is transcribed from mRNA elim<strong>in</strong>ates the bulk<br />
<strong>of</strong> the non-cod<strong>in</strong>g sequences.<br />
The differ<strong>in</strong>g expression levels <strong>of</strong> genes can be problematic when identification <strong>of</strong><br />
lowly expressed genes is desired. cDNA sequenc<strong>in</strong>g can result <strong>in</strong> many copies <strong>of</strong> the<br />
highly expressed genes be<strong>in</strong>g sequenced and few or no copies <strong>of</strong> the lowly expressed<br />
genes. One way to mitigate this problem is to normalise the expression levels <strong>of</strong> all<br />
genes <strong>in</strong> a cDNA sample by reduc<strong>in</strong>g the levels <strong>of</strong> highly expressed genes. A method<br />
developed by Zhulidov et al. (2004) and Zhulidov et al. (2005) permits the<br />
identification <strong>of</strong> rare transcripts <strong>in</strong> a cDNA population. Briefly this method utilises the<br />
specificity <strong>of</strong> duplex-specific nuclease (DSN) for double stranded DNA as a substrate.<br />
Double stranded cDNA (dsDNA) is synthesised from mRNA <strong>of</strong> <strong>in</strong>terest by us<strong>in</strong>g<br />
specific adaptors. The dsDNA is then melted and allowed to rehybridise under<br />
controlled conditions. A higher tendency <strong>of</strong> abundant transcripts to rehybridise is<br />
observed compared to rare transcripts. The dsDNA and s<strong>in</strong>gle-stranded DNA<br />
(ssDNA) population is then treated with DSN, which specifically targets dsDNA for<br />
degradation leav<strong>in</strong>g beh<strong>in</strong>d a population that has normalised levels <strong>of</strong> transcripts. PCR<br />
is then used to generate dsDNA from the ssDNA (Zhulidov et al., 2004; Zhulidov et<br />
al., 2005).<br />
4.1.2 Genome sequenc<strong>in</strong>g<br />
Genomic sequenc<strong>in</strong>g provides sequence data for all the genetic <strong>in</strong>formation conta<strong>in</strong>ed<br />
with<strong>in</strong> the cells <strong>of</strong> the organism <strong>of</strong> <strong>in</strong>terest. It is very useful <strong>in</strong> identify<strong>in</strong>g the genes<br />
conta<strong>in</strong>ed with<strong>in</strong> an organism regardless <strong>of</strong> the expression level <strong>of</strong> the gene (Bork et<br />
al., 1998). However, gene prediction becomes a challenge as cod<strong>in</strong>g regions <strong>of</strong> genes<br />
are flanked by <strong>in</strong>trons and repeats (Gilbert, 1978). The challenge <strong>in</strong> genome<br />
sequenc<strong>in</strong>g is not obta<strong>in</strong><strong>in</strong>g the sequence data itself but assembly <strong>of</strong> the data and<br />
accurate gene prediction. Most eukaryotic cells have two sources <strong>of</strong> DNA, the nucleus<br />
and the mitochondria. A larger quantity <strong>of</strong> mitochondrial DNA (mtDNA) than nucleic<br />
DNA is found <strong>in</strong> cells hence when isolat<strong>in</strong>g genomic DNA for sequenc<strong>in</strong>g and<br />
identification <strong>of</strong> nuclear genes, it is important to reduce the mtDNA content <strong>of</strong> the