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Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
Published on: February 5, 2014
RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting
Rebecca L Margraf1, Rong Mao, W Edward Highsmith
1Advanced Technology Group, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. rebecca.margraf@aruplab.com
Abstract:
Single bp mutations in the RET proto-oncogene can cause multiple endocrine neoplasia type 2 syndromes. The conventional approach for genotyping RET mutations is sequencing the exons. A closed-tube RET genotyping assay using a saturating DNA dye, unlabeled probes, and amplicon high-resolution melting analysis was developed. The method required two sequential polymerase chain reaction stages, a primary and secondary assay. The primary assay analyzed RET exons 10, 11, 13, 14, and 16 with a total of seven reactions using eight unlabeled probes. The primary assay genotyped wild-type exons, a common exon 13 polymorphism, and an exon 16 mutation, whereas other RET sequence variation was detected. The primary unlabeled probe data limited the possible genotypes for the detected RET sequence variation, which permitted genotyping in a secondary assay with only two to five reactions. Six probes were designed with the masking technique and masked selected sequence variations to allow unambiguous analysis of other mutations elsewhere under the probe. After this two-stage RET genotyping assay, less than 0.2% of exons tested would require sequencing for genotype. A blinded study generated from five wild type and 29 available RET sequence variation samples was 100% concordant with sequencing. Amplicon high-resolution melting analysis with unlabeled probes and the masking technique is a fast, accurate method for genotyping the >50 RET sequence variations.
Insights
A new closed-tube assay accurately genotypes RET proto-oncogene mutations using high-resolution melting analysis. This method significantly reduces the need for sequencing, offering a faster and more precise approach for multiple endocrine neoplasia type 2 diagnostics.
Area of Science:
- Genetics
- Molecular Biology
- Oncology
Background:
- Single base pair mutations in the RET proto-oncogene are linked to multiple endocrine neoplasia type 2 syndromes.
- Conventional RET mutation detection relies on exon sequencing, which can be time-consuming and costly.
Purpose of the Study:
- To develop and validate a novel, closed-tube assay for RET genotyping.
- To improve the efficiency and accuracy of detecting RET proto-oncogene variations.
Main Methods:
- A two-stage assay combining polymerase chain reaction (PCR) and amplicon high-resolution melting (HRM) analysis was developed.
- The assay utilized unlabeled probes and a masking technique for precise mutation detection across key RET exons.
- Unlabeled probes and masking enabled unambiguous analysis of multiple RET sequence variations.
Main Results:
- The developed assay accurately genotyped wild-type and variant RET exons, including a common exon 13 polymorphism and an exon 16 mutation.
- A blinded study demonstrated 100% concordance with sequencing for 34 samples (5 wild-type, 29 variant).
- Less than 0.2% of tested exons required confirmatory sequencing, highlighting the assay's efficiency.
Conclusions:
- Amplicon HRM analysis with unlabeled probes and the masking technique provides a fast and accurate method for genotyping over 50 RET sequence variations.
- This closed-tube assay offers a significant advancement over traditional sequencing for RET mutation analysis.
- The method is highly suitable for clinical diagnostics of multiple endocrine neoplasia type 2.
