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 o...

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Bibliographic Details
Published in:Analytical biochemistry 2003-06, Vol.317 (2), p.166-170
Main Authors: Pacey-Miller, Toni, Henry, Robert
Format: Article
Language:English
Subjects:
Alkaline phosphatase
Apyrase
Biotinylation
DNA Primers - genetics
DNA, Plant - chemistry
DNA, Plant - genetics
Exonuclease I
Genetic Variation
Hordeum - genetics
Oryza - genetics
Plants - genetics
Polymerase chain reaction
Polymorphism, Single Nucleotide - genetics
Pyrophosphate
Sequence Analysis, DNA - methods
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1016/S0003-2697(03)00089-7