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Identification of putative odorant receptors from Epiphyas postvittana 80 the coding RNA and protein (Sterky and Lundeberg, 2000) and the references within). Therefore, sequencing of cDNA, which is transcribed from mRNA eliminates the bulk of the non-coding sequences. The differing expression levels of genes can be problematic when identification of lowly expressed genes is desired. cDNA sequencing can result in many copies of the highly expressed genes being sequenced and few or no copies of the lowly expressed genes. One way to mitigate this problem is to normalise the expression levels of all genes in a cDNA sample by reducing the levels of highly expressed genes. A method developed by Zhulidov et al. (2004) and Zhulidov et al. (2005) permits the identification of rare transcripts in a cDNA population. Briefly this method utilises the specificity of duplex-specific nuclease (DSN) for double stranded DNA as a substrate. Double stranded cDNA (dsDNA) is synthesised from mRNA of interest by using specific adaptors. The dsDNA is then melted and allowed to rehybridise under controlled conditions. A higher tendency of abundant transcripts to rehybridise is observed compared to rare transcripts. The dsDNA and single-stranded DNA (ssDNA) population is then treated with DSN, which specifically targets dsDNA for degradation leaving behind a population that has normalised levels of transcripts. PCR is then used to generate dsDNA from the ssDNA (Zhulidov et al., 2004; Zhulidov et al., 2005). 4.1.2 Genome sequencing Genomic sequencing provides sequence data for all the genetic information contained within the cells of the organism of interest. It is very useful in identifying the genes contained within an organism regardless of the expression level of the gene (Bork et al., 1998). However, gene prediction becomes a challenge as coding regions of genes are flanked by introns and repeats (Gilbert, 1978). The challenge in genome sequencing is not obtaining the sequence data itself but assembly of the data and accurate gene prediction. Most eukaryotic cells have two sources of DNA, the nucleus and the mitochondria. A larger quantity of mitochondrial DNA (mtDNA) than nucleic DNA is found in cells hence when isolating genomic DNA for sequencing and identification of nuclear genes, it is important to reduce the mtDNA content of the
Identification of putative odorant receptors from Epiphyas postvittana 81 total DNA sample. Two broad approaches have been taken to sequence genomes, a shotgun approach or a clone-by-clone approach. In the shotgun approach, the genomic DNA of interest is fragmented, then size selected for library construction (Green, 1997; Weber and Myers, 1997). The ends of the DNA are end repaired followed by selection of fragments of a certain size class which are cloned into plasmid vectors and sequenced. The inserts, depending on the plasmid used can range anywhere from 2 kb to 200 kb. In the clone-by-clone approach, large overlapping clones (insert size greater than 50 kb) that cover the whole genome are constructed usually with Bacterial Artificial Chromosomes (BACs) (Olson et al., 1989; Kim et al., 1996). These BACs or libraries are then individually fragmented and sequenced by shotgun cloning, after which each BAC is assembled locally. The individual BACs are then assembled to get the genome sequence of the organism. This is helpful when assembling large eukaryotic genomes with repeats with limited computational power. 4.1.3 Recent progress in sequencing field A turning point in the sequencing field came from the development of low-cost high- throughput sequencing machines. Several sequencing platforms are now available for generating millions of bases at a fraction of the price of traditional Sanger sequencing methods, although the low cost is offset by the generation of only short read lengths, a limitation of new sequencing technologies. Intense computer power is required for the correct assembly of thousands of short reads, yet another limitation of new sequencing technologies. Of the available sequencing platforms of Roche 454 (Ronaghi et al., 1996; Dressman et al., 2003; Margulies, 2005), Illumina Genome Analyzer (Adessi, 2000; Fedurco et al., 2006; Turcatti et al., 2008), ABI/Solid (Shendure, 2005; McKernan et al., 2006) and Helicos (Braslavsky et al., 2003; Harris, 2008) the first two platforms have been used for sequencing the E. postvittana antennal transcriptome and genome respectively, and will be discussed further.
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Abstract Abstract Olfaction or the
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Acknowledgements Acknowledgements I
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Contents v Contents Abstract……
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Contents 4.2.5 Degenerate PCR .....
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List of Figures List of Figures Fig
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List of Tables List of Tables Table
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List of Abbreviations gDNA Genomic
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1.1 General overview 1 General Intr
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General Introduction 3 moth in loca
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General Introduction 5 Figure 1.1:
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General Introduction 7 1.5 Componen
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General Introduction 9 OBPs have al
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General Introduction 11 A model has
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General Introduction 15 Tanaka et a
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General Introduction 17 Table 1.1:
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General Introduction 21 et al., 200
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General Introduction 23 the attract
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General Introduction 25 leaves and
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Page 45 and 46: General Introduction 29 Expression
Page 47 and 48: General Introduction 31 BmOr2 HvOr2
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Page 51 and 52: Functional characterisation of Epip
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Concluding Discussion 131 against t
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Concluding Discussion 133 sequencin
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Appendix B 135 Buffered TB (Sambroo
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Appendix C 137 C. Appendix C - Clus
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Appendix D 139 D. Appendix D - Solu
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Appendix E 141 I II
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Appendix E 143 V
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Appendix E 145 VII continued Figure
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References 147 Ansebo, L., Ignell,
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References 149 Butenandt, A., Beckm
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References 151 Fedurco, M., Romieu,
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References 153 Große-Wilde, E., St
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References 155 Hrdy, I., F. Kuldova
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References 157 Kaissling, K. E. (19
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References 159 Krieger, J., Große-
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References 161 Lopez-Gomez, R. and
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References 163 Miura, N., Nakagawa,
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References 165 Pell, J. K., Macaula
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References 167 Røstelien, T., Stra
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References 169 Stowers, L. and Loga
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References 171 Turner, C. T., Davy,
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References 173 Vogt, R. G., Riddifo
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References 175 Widmayer, P., Heifet
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