A novel method for DNA molecular counting.
We have developed a new method for counting DNA molecules using 'capillary-plates' consisting of a large number of small glass-capillary 'channels' fused together in parallel. PCR mixtures containing serially diluted DNA templates with the DNA indicator dye Hoechst 33258 were poured into the plates and sealed with silicone rubber-plates. Following 40 PCR cycles, fluorescence microscopy revealed that the fluorescence in some channels had increased about three-times more than in others at template concentrations of 1 fM or lower. No bright fluorescence was observed in the absence of template. The relationship between the proportion of fluorescent channels in the capillary-plates and the template concentrations was linear according to Poisson probabilities in the range of 0.1-1,000 aM. These results demonstrate the amplification of single templates in the channels, and that a small number of templates could be quantified by counting the proportion of positive channels on the capillary-plates.
Hirano
K
K
Tsukuba Research Laboratory, Hamamatsu Photonics K.K., 5-9-2 Tokodai, Tsukuba City, Ibaraki 300-2635, Japan.
Fukami
T
T
D006690:Bisbenzimidazole; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D005456:Fluorescent Dyes; D004247:DNA; D006690:Bisbenzimidazole