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Tytuł oryginału: Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.
Autorzy: Fragoso Maria C. Barisson Villares, Lado Valeria S., Latronico Ana C., Frazzatto Eliana T. Salgado, Russell Alan J., Mendonca Berenice B.
Źródło: Med. Sci. Monitor 2002: 8 (1) s.BR15-BR18, il., bibliogr. 9 poz.
Sygnatura GBL: 313,278

Hasła klasyfikacyjne GBL:
  • genetyka
  • ginekologia i położnictwo
  • onkologia

    Typ dokumentu:
  • praca doświadczalna
  • tytuł obcojęzyczny

    Wskaźnik treści:
  • ludzie
  • in vitro

    Streszczenie angielskie: The identification of somatic mutations in tissues is often difficult when the number of normal alleles in the tissue far exceeds the number of mutation was not possible by direct sequencing and present a new approach that improves the identification of gsp somatic mutations. Genomic DNA was extracted from frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsŕ 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 amonunt 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. 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. 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.

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