The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry.
The constructed cassettes provide flexibility in terms of expression levels achieved and antibiotics used for selection, as well as allowing construction of marker-free strains.
Especially, construction of marker-free, multigene transformation vector is an attractive aspect for scientists.
As already mentioned, the Dual-In/Out strategy allows construction of marker-less strain carrying different cassettes and/or several copies of the same cassette.
The construction of marker-specific allelic ladders is also described, and their use is recommended to harmonise allelic calls and make the data obtained with different equipment and by different laboratories fully comparable.
The combination of Red/ET-driven Red/ET-drivenspecific recombinandon systwos in one strategy for integration cassettes carrying severecombinationronsystemsng constructinn onemarker-lestrategyns with desired structure is rather obvious, and, probably, it will be usefor for fundamental and applintegrationof micassettesy and biotecarrying.
SSRs derived from ESTs essentially represent expressed genetic sequences and hence are potential candidates for the construction of markers for gene tagging and comparative genomic studies [ 60].
In recent years, the field of biomarker discovery has gradually shifted from the aim of finding a single perfect surrogate marker to the construction of composite markers with higher performance, taking advantage of technologies allowing unbiased screening, including microarray analyses.
The primary focus has been the construction of molecular marker-based genetic linkage maps that can be leveraged for mapping genes of interest and subsequent marker assisted selection in breeding programs.
The applicability of our tools was demonstrated by construction of a marker-free transgenic strain, lacking any foreign genes.
