Elsevier

Genomics

Volume 78, Issues 1–2, November 2001, Pages 83-90
Genomics

Regular Article
High-Speed, Multicolor Fluorescent Two-Dimensional Gene Scanning

https://doi.org/10.1006/geno.2001.6649Get rights and content

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. Highthroughput 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.

References (18)

  • N.J. van Orsouw et al.

    Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis

    Genomics

    (1998)
  • A.J. Schafer et al.

    DNA variation and the future of human genetics

    Nat. Biotechnol.

    (1998)
  • J. Vijg et al.

    Screening for mutations in cancer predisposition genes

    Genetic Predisposition to Cancer

    (2001)
  • F. Sanger et al.

    DNA sequencing with chain-terminating inhibitors

    Proc. Natl. Acad. Sci. USA

    (1977)
  • C. Eng et al.

    Genetic testing: the problems and the promise

    Nat. Biotechnol.

    (1997)
  • R.S. Phelps et al.

    Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening

    Biotechniques

    (1995)
  • N.J. van Orsouw

    Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene

    Hum. Mol. Genet.

    (1996)
  • N.J. van Orsouw

    A highly accurate, low cost test for BRCA1 mutations

    J. Med. Genet.

    (1999)
There are more references available in the full text version of this article.

Cited by (15)

  • SNP discovery in associating genetic variation with human disease phenotypes

    2005, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
View all citing articles on Scopus
*

To whom correspondence and reprint requests should be addressed. Fax: (210) 562-5028. E-mail: [email protected].

View full text