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Detection of MC1R Polymorphisms with Protease-Mediated Allele-Specific Extension as an Alternative to Direct Sequencing

¹ Department of Biotechnology, The Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden;² Department of Oncology-Pathology, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden

^aaddress correspondence to this author at: Department of Biotechnology, The Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden; fax 46-8-5537-8481, e-mail joakim.lundeberg@biotech.kth.se

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The red hair color phenotype is associated with an increased risk of developing cutaneous malignant melanoma (1) and has been linked to certain variants of the gene for melanocortin-1 receptor (MC1R) (2)(3)(4). The function of the receptor is to mediate melanin production via binding of its ligand, -melanocyte-stimulating hormone, and/or adrenocorticotropic hormone (5). The presence of some MC1R variants fails to shift the production from red/yellow pigment (pheomelanin) to black/brown pigment (eumelanin), causing less efficient protection against ultraviolet radiation. The MC1R gene is highly polymorphic, and many identified variants have been associated with an increased risk of developing malignant melanoma (6)(7). Some of these variants have also been proposed to synergize with mutations of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A) to enhance the melanoma risk further (8)(9).

There are many novel technologies for the analysis of single-nucleotide polymorphisms (SNPs) and mutations. We have previously described an approach to increase the specificity of the allele-specific extension (ASE) technology for SNP genotyping and mutation detection (10). Interestingly, this allows typing of several SNPs that were not possible to analyze by unmodified ASE. The ASE technology uses the ability of DNA polymerases to distinguish matched and mismatched 3' termini of primers and thus to extend only completely matched primers. However, several reports have shown that some 3'-termini mismatches can be elongated and give false-positive results (11). This problem can be circumvented by exploiting the fact that the mismatched primers have slower reaction kinetics. The elongation can be terminated by protease (proteinase K) degradation of the polymerase before any incorrect insertions are made (10). Protease-mediated allele-specific extension (PrASE) is a flexible assay and can be performed at lower temperatures . . . [Full Text of this Article]