LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51).
However, the cost of mutation detection by DNA sequencing is high, which creates the need for adopting strategies in order to reduce cost but maintain effectiveness.
Mutation detection was performed using a range of techniques; however, all abnormal nucleotide sequences were confirmed using direct Sanger sequencing.
However, in addition to the mutation detection, the differentiation of disease causing and neutral germline mutations having no effect on the phenotype is an important but non-trivial task.
However, genetic and phenotypic heterogeneity limit mutation detection, rendering molecular diagnosis very complex.
However, at the limit of somatic mutation detection with real time PCR technologies, sequencing by Sanger is not suitable as a confirmatory method and validation of results may require a more quantitative sequence variation assay.
However, to overcome the limitation of single mutation detection, we directly sequenced mutation-specific reactions as a simple way to rapidly assess mutation associations and demonstrated that the genotypic findings were similar to that obtained by cloning virus templates.
However, a recent study indicated that a targeted mutation detection rate is approximately 27.7% in CTCs from pancreatic cancer compared with bulk cells but is negative in white blood cells [86].
Several studies concerning these genes have reported lower frequencies, however they used either protein level analysis or mutation detection and are therefore difficult to compare with microsatellite analysis.
The present data was derived from a limited number of studies, however, and may contain unintended bias based on mutation detection methods, study design or uncontrolled confounders.
