Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer.
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.
Pacey-Miller
Toni
T
Centre for Plant Conservation Genetics, Southern Cross University, Lismore 2480, Australia. tpacey@scu.edu.au
Henry
Robert
R
D019921:Biotinylation; D017931:DNA Primers / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics*; D014644:Genetic Variation; D001467:Hordeum / Q000235:genetics; D012275:Oryza / Q000235:genetics; D010944:Plants / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D018744:DNA, Plant