A SNaPshot assay for the rapid and simple detection of four common hotspot codon mutations in the PIK3CA gene

Abstract

Background: Activating mutations in the PIK3CA gene have been identified in a variety of human malignancies and are commonly detected in hotspot codons located in the helical and kinase domains in exons 9 and 20. Existing methodologies for the detection of PIK3CA mutations are time-consuming and/or expensive. In the present study we describe the first application of a PIK3CA SNaPshot assay to the screening of frequent mutations in these exons.

Findings: A SNaPshot assay for the simultaneous detection of four frequent PIK3CA hotspot mutations (E542K, E545G, E545K and H1047R) has been developed and evaluated. The assay combines multiplex PCR amplification with a multiplex primer extension assay to allow targeted detection of all four mutations in one reaction. The method was tested using samples that had previously been analysed for mutations by high-resolution melting analysis and sequencing. All mutations detected were concordant and no false positive results were obtained. Sensitivity tests showed that the SNaPshot assay could detect mutant DNA when it represents 5-10% of the total DNA present. The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.

Conclusion: The SNaPshot assay described here offers a fast, sensitive, inexpensive and specific approach to the analysis of frequent PIK3CA mutations in both fresh and archival patient samples.

Figure 1

SNaPshot detection of four common hotspot mutations in the PIK3CA gene. (A-E) SNaPshot results obtained from the analysis of bladder tumor-derived cell line DNAs illustrating (A) wildtype, (B) E542K, (C) E545G, (D) E545K and (E) H1047R electropherogram patterns. (F) SNaPshot detection of an E542K codon mutation in DNA extracted from a formalin-fixed paraffin embedded seborrheic keratosis sample. Bases are represented by the following colours: A = green; C = black; G = blue; T = red. Orange peaks (S) represent the internal Genescan-120LIZ size standards. All DNAs were extracted using a QIAmp DNA kit.

Figure 2

Sensitivity of the PIK3CA SNaPshot assay. SNaPshot could detect mutant PIK3CA when it represented 5% to 10% of the total DNA present. Representative examples for bladder tumors with (A) E542K and (B) E545K are shown.