High-speed, multicolor fluorescent two-dimensional gene scanning

Sean B. McGrath, Mangkey Bounpheng, Loyda Torres, Marco Calavetta, Charles B. Scott, Yousin Suh, David Rines, Nathalie Van Orsouw, Jan Vijg

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High-throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step - denaturing gradient gel electrophoresis - and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.

Original languageEnglish (US)
Pages (from-to)83-90
Number of pages8
JournalGenomics
Volume78
Issue number1-2
DOIs
StatePublished - Sep 1 2001

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Keywords

  • Genetic testing
  • High-voltage denaturing gradient gel electrophoresis
  • Multi-color fluorescence
  • Single-nucleotide polymorphism discovery
  • Two-dimensional DNA electrophoresis
  • Two-dimensional gene scanning

ASJC Scopus subject areas

  • Genetics

Cite this

McGrath, S. B., Bounpheng, M., Torres, L., Calavetta, M., Scott, C. B., Suh, Y., Rines, D., Van Orsouw, N., & Vijg, J. (2001). High-speed, multicolor fluorescent two-dimensional gene scanning. Genomics, 78(1-2), 83-90. https://doi.org/10.1006/geno.2001.6649