SNaPshot and CE-SSCP: Two Simple and Cost-Effective Methods to Reveal Genetic Variability Within a Virus Species
The multiplex SNaPshot and the capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) procedures are here used for rapid and high-throughput description of the molecular variability of viral populations. Both approaches are based on (1) standard amplification of genomic sequence(s), (2) labeled primers or labeled single-stranded DNA, and (3) migration of fluorescent-labeled molecules in capillary electrophoresis system. The SNaPshot technology was used to describe the diversity of 20 targeted single nucleotide polymorphisms (SNPs) selected from alignment of viral genomic sequences retrieved from public database. The CE-SSCP procedure was applied to identify the polymorphisms of two small (<500 bases in length) genomic regions of viral genomes. The different steps of SNaPshot and CE-SSCP setup procedures are presented using Potato virus Y (PVY, Potyvirus) and Plum pox virus (PPV, Potyvirus) RNA viruses as molecular targets, respectively.
Key wordsPolymorphism Genomic RNA Viruses Electrophoresis Fluorescent-labeled ss-DNA
The authors thank Alexandra Blanchard and Lucie Mieuzet (INRA, Le Rheu, France) for their help during the setup of the SNaPshot procedure, Marise Guillet (FNPPPT/INRA, Le Rheu, France) for providing the PVY-specific antibody, and Maxime Galan (CBGP, Montpellier, France) for technical advices for CE-SSCP analysis and technical support with the MegaBACE™ DNA analysis.
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