Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism

Electrophoresis
C D O'ConnellD H Atha

Abstract

We have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5--9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base deletion. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG-SSCP). Fluorescent-labeled PCR primers were used for amplification of specific exons for mutation detection. SG-SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations....Continue Reading

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Citations

Feb 9, 2002·Journal of Clinical Laboratory Analysis·Peter E BarkerCatherine D O'Connell
Sep 2, 2010·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Donald H AthaVytas Reipa
Oct 29, 2003·Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology·Catherine D O'ConnellDonald H Atha
Nov 6, 2004·Molecular Diagnosis : a Journal Devoted to the Understanding of Human Disease Through the Clinical Application of Molecular Biology·Bulbin Sunar-ReederCatherine D O'Connell
Mar 16, 2000·Diagnostic Molecular Pathology : the American Journal of Surgical Pathology, Part B·B IacopettaP Robbins

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