Cell Biology JoVE (Open Access) Citable · DOI

Pyrosequencing: A Simple Method for Accurate Genotyping

Name: Pyrosequencing: A Simple Method for Accurate Genotyping
Uploaded: 2026-08-05
Duration: 13 min 6 s
Description: Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

DOI: 10.3791/630-v

What you'll learn

✓ Prepare PCR product for pyrosequencing
✓ Run pyrosequencing reaction and data collection
✓ Use Pyrosequencing 96A1.1 software for assay setup
✓ Interpret pyrosequencing results for genotype determination

Protocol

Biopharma Insights Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Difficulty

intermediate

Total time

~3-4 hours per 96-well plate

Biosafety

BSL-1

Steps

Prepare PCR product for pyrosequencing

Clean up and prepare the PCR product to make it suitable for the pyrosequencing reaction.

▶ 02:19

Perform pyrosequencing reaction

Run the pyrosequencing reaction on the prepared sample to generate sequencing data.

▶ 06:42

Enter assay details in software

Use the Pyrosequencing 96A1.1 software to input assay-specific parameters for the run.

▶ 06:45

Run sample plate in pyrosequencer

Place the sample plate in the instrument and execute the pyrosequencing run.

▶ 08:36

Examine pyrosequencing results

Analyze the raw pyrosequencing output to determine the genotype at the target polymorphism.

▶ 11:16

💬 Comments coming soon