Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

Table 2 Benefits of the use of plasmid-free markers relative to an entire plasmid harboring a marker gene when performing reverse genetic screens.

	Distance from the inserted marker gene to the host genomic DNA	Stability of the maker primer binding sites on the integrated marker DNA	Risk of non-selectable insertions (partial plasmid sequences or incomplete marker genes)
Plasmid-free marker DNA (Library 1)	Short (as short as few bp) for both borders of the marker DNA	Stable if placed within the marker gene coding sequence	Low¹
Linearized entire plasmid marker DNA (Library 2)	Long (up to few kb) for at least one border of the marker DNA	Can be lost if placed close to the borders of the linearized vector	High²

¹Rearrangement in, or fragmentation of the introduced marker would eliminate the drug resistance selection; no transformant would be identified. ²Rearrangements and fragmentation could occur that would leave the marker gene intact, which would allow fragments of non-selectable DNA to integrate into the genome and cause secondary mutations.

ISSN: 1746-4811