METHODS: We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells.
COLD-PCR and Innovative Microarray Substrates for Detecting and Genotyping MPL Exon 10 W515 Substitutions
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2012
Abstract
METHODS: We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells.File in questo prodotto:
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