Fast and reliable screening of mutations in human tumors: Use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA)

Luisa A. Marcelino, M. Galvin, G. M. Martins, M. J. Proença, E. Mayrand, J. A. Rueff, C. J. Monteiro

Research output: Contribution to journalArticle

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Abstract

Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.

Original languageEnglish
Pages (from-to)1134-1148
Number of pages15
JournalBioTechniques
Volume26
Issue number6
DOIs
Publication statusPublished - 24 Jun 1999

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