Improving the limit of detection for Sanger sequencing: a comparison of methodologies for KRAS variant detection.

摘要: Fluorescent dye terminator Sanger sequencing (FTSS), with detection by automated capillary electrophoresis (CE), has long been regarded as the gold standard for variant detection. However, software analysis and base-calling algorithms used to detect mutations were largely optimized resequencing applications in which different alleles expected heterozygous mixtures of 50%. Increasingly, requirements are an analytic sensitivity minor A transitions from 50%-5% 5% G>C G>T transversions. In addition, we use two sample types compare limit sequence variants codons 12 13 KRAS gene between other methodologies including shifted termination assay (STA) detection, single-base extension (SBE), pyrosequencing (PS), high- resolution melt (HRM), real-time PCR (qPCR).