Colorimetric approach to high-throughput mutation analysis

Nicole E. Benoit, David Goldenberg, Shirley X. Deng, Eli Rosenbaum, Yoram Cohen, Joseph A. Califano, William H. Shackelford, Xiao B. Wang, David Sidransky

Research output: Contribution to journalArticle

Abstract

High-throughput genomic mutation screening for primary tumors has characteristically been expensive, labor-intensive, and inadequate to detect low levels of mutation in a background of wild-type signal. We present a new, combined PCR and colorimetric approach that is inexpensive, simple, and can detect the presence of 1% mutation in a background of wild-type. We compared manual dideoxy sequencing of p53 for eight lung cancer samples to a novel assay combining a primer extension step and an enzymatic colorimetric step in a 96-well plate with covalently attached oligonucleotide sequences. For every sample, we were able to detect the presence or absence of the specific mutation with a statistically significant difference between the sample optical density (OD) and the background OD, with a sensitivity and specificity of 100%. This assay is straightforward, accurate, inexpensive, and allows for rapid, high-throughput analysis of samples, making it ideal for genomic mutation or polymorphism screening studies in both clinical and research settings.

Original languageEnglish (US)
Pages (from-to)635-639
Number of pages5
JournalBioTechniques
Volume38
Issue number4
DOIs
StatePublished - Apr 2005

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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    Benoit, N. E., Goldenberg, D., Deng, S. X., Rosenbaum, E., Cohen, Y., Califano, J. A., Shackelford, W. H., Wang, X. B., & Sidransky, D. (2005). Colorimetric approach to high-throughput mutation analysis. BioTechniques, 38(4), 635-639. https://doi.org/10.2144/05384PF01