Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 XXXX Mutations.

@entity1 @entity5 @entity984 @entity406 @entity14
UNASSIGNED: The high degree of @entity5 heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance @entity984 mutations in metastatic @entity14 (mCRC) @entity1 . As a major potential source of @entity5 DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from @entity406 -fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for @entity5 genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned @entity984 mutations in mCRC @entity1 . The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC @entity1 , the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the   Cq method was used to quantitatively analyze the mutation levels associated with @entity984 in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the @entity1 analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated @entity984 gene.
