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Christine L. Baker, Cecily P. Vaughn, Wade S. Samowitz 

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1 A PIK3CA Pyrosequencing-Based Assay that Excludes Pseudogene Interference 
Christine L. Baker, Cecily P. Vaughn, Wade S. Samowitz  The Journal of Molecular Diagnostics  Volume 14, Issue 1, Pages (January 2012) DOI: /j.jmoldx Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Top panel: The reference sequence from the National Center for Biotechnology Information file NG_ Homo sapiens phosphoinositide-3-kinase, catalytic, α-polypeptide (PIK3CA) on chromosome 3. Middle panel: This is the same as the Sanger sequence of germline DNA of a PCR amplicon of exon 9 using the reverse primer specific for the gene (denoted PIK3CA sequence). Bottom panel: This sequence differs from the pseudogene sequence at codon 545, generated by sequencing an amplicon in which the pseudogene-specific reverse primer was used (denoted pseudogene sequence). The Journal of Molecular Diagnostics  , 56-60DOI: ( /j.jmoldx ) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Pyrosequencing results showing the specificity of PIK3CA amplification for codon 545. Sequences are for reverse-strand synthesis. A: Pyrogram for amplification of a sample using PIK3CA-specific primers. The sequence of PIK3CA codon 545 is GAG; thus, the reverse-strand sequence is CTC, as seen at dispensation positions 3, 5, and 6. There is no peak at dispensation 4, indicating that the pseudogene was not amplified. B: Pyrogram for amplification of a sample using pseudogene-specific primers. The sequence of the pseudogene sequence corresponding to codon 545 is GCG; thus, the reverse-strand sequence is CGC, as seen in dispensation positions 3, 4, and 6. There is no peak at dispensation 5, indicating that the gene was not amplified. The Journal of Molecular Diagnostics  , 56-60DOI: ( /j.jmoldx ) Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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