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09 January 2002

Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.

Maria C Barisson Villares Fragoso, Valeria S Lando, Ana C Latronico, Eliana T Salgado Frazzatto, Alan J Russell, Berenice B Mendonca

Med Sci Monit 2002; 8(1): BR15-18 :: ID: 420977


BACKGROUND: The identification of somatic mutations in tissues is oftendifficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We foundthat the identification of gsp mutation was not possible by direct sequencing and present a new approachthat improves the identification of gsp somatic mutations. MATERIAL/METHODS: Genomic DNA was extractedfrom frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplifiedby PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR productdid not reveal mutations, probably due to the small amount of mutant alleles. To improve this amount,the PCR products were re-amplified using as template the excised products of the mutant homoduplex andheteroduplex bands obtained at the first DGGE. RESULTS: This approach resulted in the enhancing of themutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Directsequencing of the second round PCR clearly identified the mutation R201C in the ovarian Leydig cell tumor.CONCLUSIONS: We have demonstrated a relatively rapid, convenient and reliable method to improve gsp somaticmutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutanthomoduplex bands disclosed in a first DGGE followed by direct sequencing.

Keywords: Alleles, Electrophoresis, Polyacrylamide Gel, GTP-Binding Protein alpha Subunits, Gs, Heterozygote, Mutation, Nucleic Acid Heteroduplexes, Polymerase Chain Reaction, Research Support, Non-U.S. Gov, Sequence Analysis, DNA

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Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750