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Prime Research on Biotechnology

ISSN: 2315-5299

Volume 2, Issue 2, pp. 40-46

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A simple and cost-effective method to synthesize an internal size standard amenable to use with a 5-dye system

 

Mauger S , Couceiro L, and Valero M

 

UPMC Université Paris VI, UMR7144, Laboratoire Adaptation et Diversité en Milieu Marin, Équipe BEDIM, Station Biologique de Roscoff, Place Georges Teissier, F-29682 Roscoff, France

CNRS, UMR7144, Laboratoire Adaptation et Diversité en Milieu Marin, Équipe BEDIM, Station Biologique de Roscoff, Place Georges Teissier, F-29682 Roscoff, France

 

Accepted 2nd October, 2012

 
Abstract
 

Fluorescent end-labelling 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 to detect simultaneously fragments overlapping in size by the use of different labels. However, even though multiplexing approaches are usually presented as a way to reduce genotyping costs, the price of the commercial size standards still represents a substantial deterrent. For example, a new generation of dye-systems enabling fragments labelled with up to 5 different colours is now commercially available but the price of the pertinent internal standard can easily represent 50% of the project budget. Here, we describe a simple and cost-effective method to synthesize a fluorescently labelled 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. Allele sizing using the SM594 allows a 40 fold decrease in genotyping costs and shows identical results than its commercial equivalent with maximum divergence between readings of both standards never exceeding 0.52 bp.

Keywords:
5-dye system, AFLPs, allele sizing, capillary electrophoresis, fluorescent dye, internal size standard, microsatellites
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Mauger S

Couceiro L

Valero M

 

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