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

Hurst CD, Zuiverloon TC, Hafner C, Zwarthoff EC, Knowles MA - BMC Res Notes (2009)

Bottom Line: 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.The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK. c.hurst@leeds.ac.uk

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Finally, we assessed the sensitivity of the SNaPshot assay by mixing heterozygous mutant DNA from low grade/stage bladder tumors (that are commonly diploid or near-diploid) with normal DNA in varying proportions. We were able to detect mutations E542K, E545K and H1047R when mutant DNA represented 5–10% of the total input DNA. Figure 2 shows representative examples of results obtained for codons E542K and E545K. As mutant DNAs used in these experiments were heterozygous, the observed sensitivity results equate to being able to detect one mutant allele in a background of 39 (5%) or 19 (10%) wildtype alleles. This level of detection is comparable to that reported for the FGFR3 SNaPshot assay of van Oers et al. [17]. We were also able to achieve a 5% level of detection for codon E545K when mutant DNAs from two triploid bladder tumor cell lines (TCC-SUP and BFTC909) were used (data not shown). Calling the presence of low-level mutations is more difficult and in these cases more than one individual should perform scoring and the assay should be repeated to confirm that the mutations do not represent artefacts.


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

Hurst CD, Zuiverloon TC, Hafner C, Zwarthoff EC, Knowles MA - BMC Res Notes (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683860&req=5

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.
Mentions: Finally, we assessed the sensitivity of the SNaPshot assay by mixing heterozygous mutant DNA from low grade/stage bladder tumors (that are commonly diploid or near-diploid) with normal DNA in varying proportions. We were able to detect mutations E542K, E545K and H1047R when mutant DNA represented 5–10% of the total input DNA. Figure 2 shows representative examples of results obtained for codons E542K and E545K. As mutant DNAs used in these experiments were heterozygous, the observed sensitivity results equate to being able to detect one mutant allele in a background of 39 (5%) or 19 (10%) wildtype alleles. This level of detection is comparable to that reported for the FGFR3 SNaPshot assay of van Oers et al. [17]. We were also able to achieve a 5% level of detection for codon E545K when mutant DNAs from two triploid bladder tumor cell lines (TCC-SUP and BFTC909) were used (data not shown). Calling the presence of low-level mutations is more difficult and in these cases more than one individual should perform scoring and the assay should be repeated to confirm that the mutations do not represent artefacts.

Bottom Line: 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.The application of the method to the analysis of DNAs extracted from formalin-fixed paraffin-embedded samples was also demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Research UK Clinical Centre, Leeds Institute of Molecular Medicine, St James's University Hospital, Leeds, UK. c.hurst@leeds.ac.uk

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.

No MeSH data available.


Related in: MedlinePlus