Hybridization detection method
A detection method and a technology of a reaction field, which are applied in the field of hybridization detection, can solve problems such as noise fluorescence, hybridization detection accuracy may not be sufficient, etc., and achieve the effect of improving detection accuracy
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Embodiment 1
[0121] In Example 1, it was investigated whether mishybridization can be reduced and the S / N ratio of hybridization can be improved by performing a high-temperature treatment step after hybridization. The outline of the experimental procedure is as follows.
[0122] First make the bottom plate used in the experiment. The silicon bottom plate cut into 1 cm square was washed, and after the surface was cleaned and hydroxylated by ozone treatment and hydrogen peroxide treatment, it was heated and dried at 120° C. for 30 minutes. Then, add aminopropylsilane to toluene at a volume ratio of 2% relative to toluene to prepare a solution, and immerse the washed bottom plate in the solution at 50° C. for 30 minutes to make the hydroxyl groups on the silicon surface and aminopropyl groups The silane was reacted and washed with toluene and water. Then, 100 mg of succinic anhydride was dissolved in 10 ml of dimethylformamide to prepare a solution, and the bottom plate was immersed in the ...
Embodiment 2
[0129] In Example 2, similarly to Example 1, it was investigated whether mishybridization could be reduced and the S / N ratio of hybridization could be improved by performing a temperature raising treatment step after hybridization. In addition, in this experiment, the fluorescence intensity at the time of hybridization detection was measured by binding the fluorescent dye Cy3 to the sample nucleic acid.
[0130] The outline of the experimental procedure is as follows.
[0131] First, avidin is immobilized on the bottom plate surface. Immerse the jeansilicon (registered trademark, manufactured by Toyo Steel Co., Ltd.) in 300 μl (100 mg / l, HEPES buffer) avidin solution at room temperature for 30 minutes, fix the avidin, wash with HEPES Tween Buffer for 5 minutes, Three buffer exchanges were performed with HEPESBuffer.
[0132] Probe fixation is then performed. The DNA oligomer was treated with biotin in advance to prepare the probe immobilization solution, and then immersed t...
Embodiment 3
[0138] In Example 3, it was investigated whether fluorescence noise (background noise) can be reduced and the S / N ratio of hybridization can be improved by adding an intercalator that binds to a double-stranded nucleic acid and then performing a temperature-raising step.
[0139] The outline of the experimental procedure is as follows. In the hybridization buffer, add 150nM DNA oligomer (probe nucleic acid strand), 150nM DNA oligomer complementary or non-complementary to the DNA oligomer (target nucleic acid, and non-target sample nucleic acid), intercalator ( SYBR (registered trademark) Green I), preparation solution. Then, the solution was heated at 95°C for 5 minutes to denature the nucleic acid into a single strand, and then rapidly cooled to 4°C. Then, after incubation at 25°C for 1 minute, the fluorescence intensity generated by the intercalator was detected. Then, the temperature was raised by 1° C. and incubated at each temperature for 1 minute, and then the fluoresc...
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