Fluorescence energy transfer detection as a homogeneous DNA diagnostic method

Abstract

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.

Figure 1

The TDI assay.

Figure 2

Real-time fluorescence detection in the TDI assay for the cystic fibrosis ΔF508 mutation. The average fluorescence emissions of the three dye species for each cycle during the primer extension reaction are plotted against the cycle number. Blue diamonds = fluorescein emission, magenta circles = ROX emission, green triangles = TAMRA emission. (A) Normal individual showed progressive quenching of fluorescein emission with concomitant enhancement of ROX emission (initial slope = 136) but no significant change in TAMRA emission, signifying that ROX-ddC was the only nucleotide incorporated. (B) Affected individual showed quenching of fluorescein and enhancement of TAMRA (initial slope = 118) emission but no significant change in ROX emission, signifying that only TAMRA-ddU was incorporated. (C) Carrier showed quenching of fluorescein and enhancement of both ROX and TAMRA emissions (initial slopes of 72 and 51, respectively), signifying that both ROX-ddC and TAMRA-ddU were incorporated. (D) No significant change in fluorescence emission was seen for any of the three dyes because neither ROX-ddC nor TAMRA-ddU were incorporated. The slopes in the heterozygote are about half of those for the homozygotes.

Figure 3

Detection of known mutations by the TDI assay. The initial slope of change in fluorescence intensity of ROX and TAMRA are plotted against each other. (A) Thirty-eight individuals were tested for the cystic fibrosis ΔF508 mutation (with four salmon sperm DNA negative controls), where ROX-ddC and TAMRA-ddU were used in the reaction for the normal and mutant alleles, respectively. The data points clustered into four groups, which represented the normal C/C (green triangles), affected T/T (red squares), and carrier C/T (blue diamonds) populations in addition to the control samples (open magenta circles) where no dye-terminators were incorporated. (B) Forty-eight individuals (and 3 negative controls) were tested for the hemochromatosis C282Y mutation, where ROX-ddG and TAMRA-ddA were used for the normal and mutant alleles, respectively. The data points clustered into the normal G/G (green triangles), affected A/A (red squares), carrier G/A (blue diamonds) populations. One individual clusters with the negative controls (open magenta circles) because the PCR assay failed (solid magenta circle). (C) Twenty-nine individuals (and 3 negative controls) were tested for the autosomal-dominant MEN C634F mutation, where TAMRA-ddG and ROX-ddU were used for the normal and mutant alleles, respectively. The data points segregated into just the normal G/G (red squares) and affected heterozygous G/T (blue diamonds) groups. One sample had slightly positive slopes of intensity change for both ROX and TAMRA (open blue diamond) due to poor PCR yield whose genotype was deemed indeterminate.