Abstract
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.
Keywords: SNP, Pyrosequencing, M13 tail, Marker-assisted selection, Cost-effective
Materials and Reagents
Equipment
Software
Procedure
Notes
Recipes
Acknowledgments
This protocol is adapted from Silvar et al. (2011). The pyrosequencing assay, including annealing plate preparation, immobilization of PCR products to streptavidin beads and the preparation of single-stranded pyrosequencing template DNA were basically carried out as described in the manufacturer´s instructions.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.