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

Biotechniques. 1999 Jun;26(6):1134-8, 1140-2, 1144 passim. doi: 10.2144/99266rr01.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Biopsy
  • CHO Cells
  • Color
  • Cricetinae
  • DNA Mutational Analysis / methods*
  • DNA Primers
  • DNA, Neoplasm / analysis
  • DNA, Neoplasm / genetics*
  • Exons
  • Fluorescent Dyes
  • Genes, ras / genetics
  • Genetic Testing / methods*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / pathology
  • Molecular Sequence Data
  • Point Mutation*
  • Polymorphism, Single-Stranded Conformational
  • Stomach Neoplasms / genetics
  • Stomach Neoplasms / pathology

Substances

  • DNA Primers
  • DNA, Neoplasm
  • Fluorescent Dyes