Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.
BACKGROUND
The identification of somatic mutations in tissues is often difficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We found that the identification of gsp mutation was not possible by direct sequencing and present a new approach that improves the identification of gsp somatic mutations.


METHODS
Genomic DNA was extracted from frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplified by PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR product did 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 and heteroduplex bands obtained at the first DGGE.


RESULTS
This approach resulted in the enhancing of the mutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Direct sequencing 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 somatic mutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutant homoduplex bands disclosed in a first DGGE followed by direct sequencing.
Fragoso
Maria C Barisson Villares
MC
Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular, LIM 42, Hospital das Clínicas, University of Sao Paulo School of Medicine, Sao Paulo, Brasil.
Lando
Valeria S
VS
Latronico
Ana C
AC
Frazzatto
Eliana T Salgado
ET
Russell
Alan J
AJ
Mendonca
Berenice B
BB
D000483:Alleles; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D010051:Ovarian Neoplasms / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D009692:Nucleic Acid Heteroduplexes; D019205:GTP-Binding Protein alpha Subunits, Gs