Fast and reliable screening of mutations in human tumors: use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA).
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.
Marcelino
L A
LA
Faculty of Medical Sciences UNL, Lisbon, Portugal.
Galvin
M
M
Martins
G M
GM
Proença
M J
MJ
Mayrand
E
E
Rueff
J A
JA
Monteiro
C J
CJ
D000818:Animals; D001483:Base Sequence; D001706:Biopsy; D016466:CHO Cells; D003116:Color; D006224:Cricetinae; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D005091:Exons; D005456:Fluorescent Dyes; D011905:Genes, ras / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology; D008969:Molecular Sequence Data; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology
D017931:DNA Primers; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes