Single-nucleotide polymorphism detection in plants using a single-stranded gyrosequencing protocol with a universal biotinylated primer'
Pacey-Miller, T & Henry, RJ 2003, 'Single-nucleotide polymorphism detection in plants using a single-stranded gyrosequencing protocol with a universal biotinylated primer', Analytical Biochemistry, vol. 317, no. 2, pp. 165-170.
Forest Ecology and Management journal home page available at http://www.elsevier.com/
Publisher's version of article available at http://dx.doi.org/10.1016/S0003-2697(03)00089-7
Analysis of variations in plant genomes is increasingly focused on single-nucleotide polymorphism (SNP) analysis, increasing the need for fast yet reliable, simple, and cost-effective techniques to handle the large number of these polymorphisms within large plant genomes. Pyrosequencing technology offers a technique that takes advantage of the interaction of four enzymes in a single-tube assay to measure DNA synthesis in real time. Pyrosequencing provides a DNA sequence and an advantage over alternative techniques in poorly characterized genomes such as those of most plant species. Here we compare the use of both single-stranded and double-stranded template Pyrosequencing on plant tissue for SNP identification. Different enzymatic strategies for double-stranded template preparation were compared. Preparation of double-stranded template from plant tissue required labor-intensive purification to allow double-stranded Pyrosequencing. A more cost-effective and less labor-intensive alternative to double-stranded template preparation in plants has been developed using a universal biotinylated primer to improve the efficiency of single-stranded Pyrosequencing. This provides an efficient high-throughput method for SNP analysis by Pyrosequencing.