A rapid fluorescence based multiplex polymerase chain reaction--single-strand conformation polymorphism method for p53 mutation detection.
We have developed a fast single-strand conformation polymorphism (SSCP) technique to screen for mutations and polymorphisms in exons 5-8 of the human tumor suppressor gene p53. We use multiplex polymerase chain reaction (PCR) to amplify the four exons in one single PCR reaction and then fluorescent SSCP for screening. p53 fragments are labeled with three different colors and a fourth color is used for an internal size marker calibrating the gel. The method was evaluated in two ways: (i) 16 different cell lines with known mutations were tested blindly for band-shifts with SSCP, and (ii) 32 human urinary bladder cancer samples were screened for mutations using the present technique. After screening for mutations all exons from all samples were sequenced, both sense as well as antisense strands. Evaluating the method with four different gels shows that 21/23 mutations and polymorphism were detected in the cell lines and that 10/10 mutations and polymorphisms were detected in the patient samples. Sensitivity, specificity, positive and negative predictive values were 91/100%, 88/ 97%. 78/77% and 96/100% for cell lines / patient samples, respectively. Sensitivity, using one SSCP gel only, was 87% (20/23) for cell lines and 90% (9/10) for patient samples. We conclude that our modified SSCP technique is efficient and has a sensitivity close to 100% in detecting mutations.
Department of Biosciences at NOVUM, Karolinska Institute, Huddinge, Sweden. petra.berggren@cnt.ki.se
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Berggren
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Steineck
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Hemminki
D004252:DNA Mutational Analysis; D005091:Exons; D005456:Fluorescent Dyes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D005456:Fluorescent Dyes; D016159:Tumor Suppressor Protein p53