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Characterization of complex DNA modifications underlying gene expression patterns is based on quantifiable data. Pyrosequencing is a sequence-based detection technology that enables rapid and accurate quantification of sequence variation. Streamlined protocols, analysis flexibility, and elegant output make Pyrosequencing technology a highly adaptable tool for research in a broad range of disciplines.
See how elegant chemistry and sensitive technology enable quantification of sequence variation. PyroMark platforms, Pyrosequencing reaction cascade. Learn more. Pyrosequencing Applications Pyrosequencing Applications. Discover new possibilities with Pyrosequencing-based detection and quantification.
Pyrosequencing Protocols | Sharon Marsh | Springer
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Potato Germplasm Enhancement Laboratory
PyroMark instrument methods and pulse time settings. Anna M Casas.
Ernesto Igartua. Frank Ordon. Original research article A brief version of this protocol appeared in:.
Similar Protocols. Reproducibility Feedback. Share your feedback. 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. The amplicon size should be between and bp. The tailed primer forward or reverse should not be longer than 20 bp. The sequencing primer should be designed between one and five nucleotides of the target SNP.
Select those primers with the highest scores assigned by the software according to their quality for PCR and Pyrosequencing. Even if other DNA source is considered e. PCR product , a minimum of 50 ng should be used as well. At this step, biotin is being incorporated into the PCR product, as described by Schuelke for fluorescent labelling of PCR fragments. Check the presence of a single and strong DNA fragment with the expected size. If there is any unspecific product, which is highly unusual if the primer design was performed accurately, a HotStart Taq polymerase might be used with the same PCR mix described above, except for MgCl 2 2 mM , dNTPs 0.
Before starting the pyrosequencing assay, let the buffers reach room temperature.
Seal the plate and shake the PCR plate constantly at ca. Place the vacuum prep tool VPT in the water trough and switch on the vacuum pump Figure 1. Figure 1. Vacuum workstation with the vacuum prep tool VPT and the different troughs Flush the filter probes for 20 sec.
Denaturation solution for 5 sec. Washing buffer for 5 sec. Switch off the vacuum pump and return the VPT the horizontal position. Shake the VPT gently and let it rest for a few seconds on the bottom of the wells. Move the VPT to the water trough to wash the filters by agitating for 10 sec. Cool the PSQ plate at room temperature for at least 5 min to allow the annealing of the sequencing primer. Load the cartridge with the estimated amount of PSQ reagents.
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Place the plate in the PSQ 96 instrument and insert the cartridge carefully. Figure 2. The tailing of reverse primers should work in the same efficient way.