Homologous recombination can be used in the lab to replace a gene of interest with a selection marker. To confirm that the selec
tion marker is integrated into the correct place in the genome, sometimes a negative selection is used in addition to the standard positive selection marker. Which of the following would act as a negative selection marker? a. Transformation of a different gene, which does not act as a selection marker, to replace the gene of interest.â
b. A second marker in the knock out cassette, that if inserted into the genome results in cell death when plated on selective media.
c. Transforming the cells with two different knock out cassettes, each containing the same selection marker3. â
b. A second marker in the knock-out cassette, that if inserted into the genome results in cell death when plated on selective media.
Explanation:
General recombination, also known as homologous recombination, refers to the naturally occurring process of exchange of genetic material between pairs of homologous DNA sequences. This process (homologous recombination) can be exploited by genetic engineering to insert DNA segments of interest at target genes. Moreover, a cassette is a mobile DNA segment containing almost a gene and a recombination site, which is integrated into the <em>locus</em>/<em>loci</em> of interest by homologous recombination. A cassette may contain a DNA segment called 'negative marker' which prevents growth under particular conditions, while a positive marker permits growth under certain conditions. In consequence, a second marker consisting of a drug cassette may be used as a negative marker in order to evidence its insertion by inducing cell death when they are plated in selective conditions.
The cell wall of oomycetes, however, is not composed of chitin, as in the fungi, but is made up of a mix of cellulosic compounds and glycan. The nuclei within the filaments are diploid, with two sets of genetic information, not haploid as in the fungi.
