Mechanisms of Olfaction in Insects - ResearchSpace@Auckland ...

Identification of putative odorant receptors from Epiphyas postvittana 92
CCCGGTAAAATTAAAATATAAACTTC-3‟, specific for the cytochrome oxidase I
gene of the mitochondrial genome of E. postvittana, were used in a PCR reaction to
test for mtDNA contamination in the gDNA sample. Primers for Takeout 3, which is a
nuclear gene, were used for a direct comparison of the nuclear versus mtDNA (5‟-
AAAGGCGAGGGTCACTACAAG-3‟ TO3 forward primer and 5‟-
GGTTCTTCAGTGCGTACACCT-3‟ TO3 reverse primer). The PCR reaction mix
was prepared and the first round of PCR conducted, as stated in section 4.2.5. The
products were analysed on 1% agarose gel with 1x SYBR safe DNA gel stain
(Invitrogen) and visualised on the ImageQuant 300 system (GE Healthcare Life
Sciences). One lane of 75 bp paired-end genome sequencing was done as stated in
section 4.2.10 and Bowtie, a short read aligner (Langmead et al., 2009) was used for
mapping mitochondrial sequences in GenBank and a 2,216 base pair region of the E.
postvittana mitochondrial genome containing the cytochrome oxidase I and II genes
to the 13 million paired-end reads obtained from one lane of Illumina sequencing.
4.2.12 qRT-PCR primer design
Primers were designed using Oligo Explorer 1.1.0 (Kuulasmaa, 2000) and where
possible primers were designed using the following stringent conditions: optimal
temperature between 50-60°C, GC% content between 40-60%, and at least 3 GC
dinucleotides at the 3‟end for primer stability. The primers were between 20–23 bp in
length and the amplicon size was between 100–200 bp. The primers were made such
that the formation of primer dimers are minimised and the primers do not self anneal.
The primer pairs for the candidate OR sequences identified from the microarray
screening and transcriptome sequencing are given in Table 4.2.