Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.
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