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Illumina sequencing involves the incorporation of fluorescent nucleotides into DNA that distinguish A, C, T, and G.
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- *9.18 Comparative genomics offers insights into the relationship between homologous genes and the organization of genomes. When the genome of C. elegans was sequenced, it was striking that some types of sequences were distributed nonrandomly. Consider the data obtained for chromosome V and the X chromosome shownbelow. The following figure shows the distribution of genes, the distribution of inverted and tandem repeat sequences, and conserved genes (the location of transcribed sequences in C. elegans that are highly similar toyeast genes).a. How do the distributions of genes, inverted and tandemrepeat sequences, and conserved genes compare?b. Based on your analysis in (a), what might you hypothesizeabout the different rates of DNA evolution (change) on the arms and central regions of autosomesin C. elegans?c. Curiously, meiotic recombination (crossing-over, discussed in Chapter 12, p. 333) is higher on the arms of autosomes, with demarcations between regions of high and low crossing-over at the boundaries between conserved and nonconserved genes seen in the physical map. Does this information support your hypothesis in (b)?
- You just sequenced a single genome sample using Illumina and you received two files with your data from the sequencing core. Was the sequencing mostly likely single-end or paired-end?
- 9.12 Describe the steps you would take to obtain a null allele in your favorite yeast gene (YFG) using homologous recombination if you have available a yeast strain that is sensitive to the antibiotic kanamycin, pBluescript II plasmids (see Chapter 8, p. 176) with the DNA inserts diagrammed in Figure 9.B, and are able to transform yeast with a targeting vector, once you construct it. In Figure 9.B, EcoRI, HaeII, HindIII, and PstI are restriction enzymes (see Chapter 8, p. 174) that cleave these DNAs at the sites shown, and the distances between the sites aregiven in kb. As part of your answer, diagram the targeting vector you would construct and the structure of the chromosomal region once YFG is knocked out using this targeting vector. Also, describe how you would use PCR to confirm that you had obtained a null allele at the gene, and indicate on your diagrams the regions you would use for designing PCR primers. Remember that the absence of a PCR product does not provide strong evidence for a specific DNA arrangement, as a PCR could fail for any number of reasons.