RET Proto-Oncogene Genotyping Using Unlabeled Probes, the Masking Technique, and Amplicon High-Resolution Melting Analysis

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.

LightCycler Technology in Molecular Diagnostics

“…Unlabeled probe designs have been implemented in the clinical laboratory for F5 and F2 assays. 33,34 Unlabeled probe genotyping has also been reported for RET, 35 HSV typing, 36 and common variants of CFTR, 34 ACVRL1 and ENG. 37 …”

“…Such a combined analysis, in combination with masking common polymorphisms, can be used to efficiently identify mutations in multiple endocrine neoplasia type II. 35 …”

Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

“…The feasibility of duplex amplicon genotyping (3 ) with internal temperature calibrators (15 ) has previously been demonstrated. In the present study, we demonstrate the genotyping accuracy of a quadruplex high-resolution amplicon-melting assay for mutations in F5 5 [coagulation factor V (proaccelerin, labile factor)] (1691GϾA), F2 [coagulation factor II (thrombin)] (20210GϾA), and MTHFR (1298AϾC and 677CϾT) genes.…”

“…The use of saturating DNA dyes and high-resolution melting analysis provides some attractive solutions for genotyping (3)(4)(5)(6)(7)(8). For example, unlabeled probes (simple oligonucleotides) can be used to genotype single-base variants and small insertions or deletions (3,(5)(6)(7)(8)(9)(10)(11)(12), and the genotype is assigned by the probe's characteristic melting temperature (T m ) 4 .…”

“…For example, unlabeled probes (simple oligonucleotides) can be used to genotype single-base variants and small insertions or deletions (3,(5)(6)(7)(8)(9)(10)(11)(12), and the genotype is assigned by the probe's characteristic melting temperature (T m ) 4 . Even simpler conceptually is high-resolution analysis of amplicon melting, which uses only PCR primers (3,4 ).…”