کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2199975 | 1551181 | 2008 | 4 صفحه PDF | دانلود رایگان |
Detection of single nucleotide polymorphisms (SNPs) and of mutations is of importance in the field of genetics, biomedical research and in vitro diagnosis. We report here a genotyping procedure that can be virtually applied to any locus within a genome: it uses α-phosphorothioate deoxynucleotides in a primer-extension step followed by an ExoIII treatment. Non-extended primers are hydrolyzed whereas extended primers resist this treatment, indicating which nucleotide has been incorporated, i.e. the genotype of the locus. A 3-bp deletion in the CFTR gene (F508del, the most prevalent mutation involved in cystic fibrosis) was used as a model, in a single-tube procedure for each nucleotide to be tested. Human genomic DNA samples were correctly genotyped in less than 3 h by a solid-phase PCR followed by primer extension, ExoIII treatment and an ELISA-like detection method. The same principle (primer extension with α-phosphorothioate deoxynucleotide, ExoIII treatment) should also be combined with other detection systems such as gel or capillary electrophoresis, mass spectrometry or DNA chips.
Journal: Molecular and Cellular Probes - Volume 22, Issues 5–6, October–December 2008, Pages 320–323