Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer.

Toni Pacey-Miller; Robert Henry

doi:10.1016/s0003-2697(03)00089-7

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Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer.

Toni Pacey-Miller·Robert Henry

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Abstract

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.

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Biotinylation DNA Primers DNA, Plant Genetic Variation Hordeum Oryza Plants Polymorphism, Single Nucleotide Sequence Analysis, DNA Comparative Study Journal Article Research Support, Non-U.S. Gov't

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Toni Pacey-Miller, Robert Henry. 2003-06-15. Single-nucleotide polymorphism detection in plants using a single-stranded pyrosequencing protocol with a universal biotinylated primer.. https://doi.org/10.1016/s0003-2697(03)00089-7

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