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. 2016 Nov 29;107(11):1677–1686. doi: 10.1111/cas.13073

Figure 4.

Figure 4

Gene testing of subject No. 7. (a) Immunochemistry (IHC) using four antibodies to mismatch repair (MMR) proteins. Note the loss of PMS2 protein expression in cancer cell nuclei. (b) RT‐PCR/direct sequencing analysis of the PMS2 gene in subject No. 7. The upper panel indicates the sequencing profile showing the c.241G>T (p.Glu81Stop) mutation from a puromycin‐treated total RNA sample and the middle panel shows the sequencing profile without the puromycin treatment. Note that the signal ratio of the mutated allele to the wild‐type allele increased in the absence of the puromycin treatment and the c.241G>T (p.Glu81Stop) mutation appeared to be a homozygous mutation. (c) Long/nested PCR/direct sequencing analysis of the PMS2 gene in subject No. 7. An analysis of the genomic DNA by a long PCR/nested PCR/direct sequencing analysis showed the c.241G>T mutation (upper panel) without the presence of the wild type allele as compared to that of the control (middle panel). Equal amounts of the genomic DNA from subject no. 7 and the healthy control was mixed and subjected to the long PCR/nested PCR/direct sequencing analysis. This experiment resulted in the DNA sequence looking like the heterozygous mutation (lower panel), supporting the reliability of the homozygous mutation detected in subject No. 7.