A Simple and Cost-Effective Method to Synthesize an Internal Size Standard Amenable to Use with a 5-Dye System
Résumé
Fluorescent end-labeling joint to capillary electrophoresis has become a common practice for PCR fragment analysis. Besides its sensitivity/reproducibility, the major advantage of this method is the possibility of detect simultaneously fragments overlapping in size by the use of different labels. However, even thought multiplexing approaches are usually presented as a way to reduce genotyping costs, the price of the commercial size standards still represents a substantial deterrent. As an example, a new generation of dye-systems allowing fragments labeled up to 5 different colors is now commercially available but the price of the pertinent internal standard can easily represent the 50% of the project budget. Here, we describe a simple and cost-effective method to synthesize a fluorescently labeled size standard amenable to use with a 5-dye system, the SM594. Its performance in fragment size determination is demonstrated over a total of 63 alleles covering different molecular markers, species, and dyes. In summary, allele calling using the SM594 and the equivalent commercial standard shows identical results and maximum divergence between lectures from both standards never exceed 0.64 bp. Since genotyping costs can be reduced by a 40 factor, the use of the SM594 might allow researchers to address projects covering more species/individuals/loci.
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