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

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 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 the template-directed dye-terminator incorporation assay, is shown to be highly sensitive and specific and is suitable for automated genotyping of large numbers of samples.

Family-based association study of 76 candidate genes in bipolar disorder: BDNF is a potential risk locus

“…Genotyping was performed by either single-base extension with fluorescent resonance energy transfer (SBE-FRET), 36 single-base extension with fluorescence polarization (SBE-FP) 37 using a modified protocol as described previously, 22 or length-multiplexed singlebase extension (LM-SBE) 38 with the following modifications. From 16-18 individual PCR amplicons were multiplexed rather than 50 amplicons.…”

Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes

“…The distinctive biomolecular recognition and signal transduction capabilities of molecular beacons (MB) (Tyagi and Kramer 1996) have increasingly been applied to many medical diagnosis applications (Chen and Kwok 1997;Piatek et al 1998) and biological assays, including genotyping for SNP (Kwok 2001;Syvanen 2001), multiplex genetic analysis (Marras et al 1999;Vet et al 1999), quantitative PCR (Vogelstein and Kinzler 1999;Chen et al 2000), enzymatic reaction of proteins (Fang et al 2000;Tung et al 2000), and detection of mRNA in living cells (Sokol et al 1998;Tsuji et al 2000;Fang et al 2002). MB is a class of fluorescence resonance energy transfer (FRET) molecules that are synthesized in a hairpin structure with stem and loop portions.…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…The second is to stitch two fluorescent groups together from distant spaces to promote energy transfer between the two fluorophores. 15,70 To apply FRET principle for SNP typing, the reactions can be devised so that the formation of allele-specific products is correlated to the occurrence or disappearance of FRET phenomenon. Once a direct correlation is established between the reactions and FRET, monitoring of FRET during the allele discrimination reactions can be used to infer the formation of allele-specific products and thus to identify the genotypes of a sample.…”