Template-directed dye-terminator incorporation (TDI) assay: a homogeneous DNA diagnostic method based on fluorescence resonance energy transfer

Xiangning Chen* and Pui-Yan Kwok

Division of Dermatology, Washington University School of Medicine, 660 S. Euclid Ave, Box 8123, St. Louis, MO 63110, USA

Received September 17, revised and accepted November 13, 1996

*To whom correspondence should be addressed. E-mail: sam@psts.wustl.edu

ABSTRACT

A new method for DNA diagnostics based on template-directed primer extension and detection by fluorescence resonance energy transfer is described. In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5'-fluorescein labeled primer (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of the allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq DNA polymerase (Klentaq1-FY). The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method, which we call template-directed dye-terminator incorporation (TDI) assay, is shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples.

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Last revised: January 13, 2004.