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Nucleic Acids Research, Vol 26, Issue 10 2398-2406, Copyright © 1998 by Oxford University Press

ARTICLES

Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests

NJ van Orsouw, RK Dhanda, RD Rines, WM Smith, I Sigalas, C Eng and J Vijg
Molecular Genetics Section, Gerontology Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, Suite 921, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. nvanorso@bidmc.harvard.edR

With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non- denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.
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