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Updated: Aug 29, 2026
Pyrosequencing: A Simple Method for Accurate Genotyping
Published on: January 8, 2008
Pyrosequencing technology as a method for the diagnosis of multiple endocrine neoplasia type 2
Kent E Kruckeberg1, Stephen N Thibodeau
1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.
Background:
Multiple endocrine neoplasia type 2 (MEN2) is a cancer syndrome with well-characterized causative mutations. Missense mutations in approximately 15 codons of the RET gene have been linked to disease phenotypes in the vast majority of cases. These missense mutations range from very simple single nucleotide base changes to more numerous changes at a given codon; they therefore are often tested for by more than one DNA-based diagnostic method. We developed and evaluated a Pyrosequencing technology-based approach for MEN2 mutation testing that allows both simple and complex mutations to be analyzed on one platform.
Methods:
Archived DNA from peripheral blood of patients referred to the Mayo Clinic Molecular Genetics laboratory for MEN2 testing was selected. One to all of codons 609, 611, 618, 620, 630, 634, 768, 804, and 918 were analyzed by Pyrosequencing technology to match the original analysis of each patient. Template PCRs were set up using an automated liquid handler; the subsequent post-PCR preparation step was performed manually, and the sequencing was performed by a PSQ 96 instrument. Samples were tested in batch sizes expected to occur routinely.
Results:
We analyzed samples from 217 patients who previously tested negative for MEN2 and 230 patients who previously tested positive, for a total of 1449 sequencing reactions. One discrepant result was found (100% concordant for negatives and 99.6% concordant for positives). A total of 37 unique mutations or alterations of unknown significance were analyzed.
Conclusion:
Pyrosequencing technology offers an accurate, nonisotopic, simple, and rapid method for the analysis of DNA from patients suspected of having MEN2.
Insights
Pyrosequencing accurately detects Multiple Endocrine Neoplasia type 2 (MEN2) gene mutations. This rapid DNA analysis method is effective for diagnosing this cancer syndrome.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Multiple Endocrine Neoplasia type 2 (MEN2) is a hereditary cancer syndrome.
- Causative mutations are primarily missense mutations in the RET gene.
- Existing diagnostic methods may require multiple DNA-based approaches for comprehensive mutation analysis.
Purpose of the Study:
- To develop and evaluate a Pyrosequencing-based method for MEN2 mutation testing.
- To assess the platform's capability for analyzing both simple and complex RET gene mutations.
- To provide an efficient diagnostic tool for MEN2.
Main Methods:
- Analysis of archived DNA from patients with suspected MEN2.
- Targeted sequencing of specific RET gene codons (609, 611, 618, 620, 630, 634, 768, 804, 918) using Pyrosequencing.
- Utilized automated liquid handling for PCR setup and a PSQ 96 instrument for sequencing.
Main Results:
- 1449 sequencing reactions were performed on 447 patients (217 negative, 230 positive for MEN2).
- Achieved 100% concordance for negative and 99.6% concordance for positive results.
- Successfully analyzed 37 unique mutations, including alterations of unknown significance.
Conclusions:
- Pyrosequencing technology provides an accurate and rapid method for MEN2 DNA analysis.
- The platform is suitable for detecting a wide range of RET gene mutations.
- This nonisotopic method simplifies and accelerates MEN2 mutation testing.
