The DNA chip hybridization pattern was determined by detecting the marker signal. The detection methods of fluorescence labeled hybridization signal are the most used by the researchers, so the corresponding detection methods are the most mature.
Sample preparation includes sample separation, purification, amplification and labeling.
First, the nucleic acid, DNA or mRNA of the sample is separated and purified from the tissue cells by conventional methods. Due to the limited sensitivity of the current chip detection instrument, the target sequence of the sample needs to be amplified efficiently and specifically. The samples were labeled mainly by fluorescence, but also by biotin and radionuclide.
2. Molecular hybridization
After the sample is amplified and labeled, it can be hybridized with the probe array on the DNA chip. Chip hybridization is similar to traditional hybridization methods such as Southern blot, which belongs to solid-liquid hybridization. The probe molecules are fixed on the surface of the chip and react with the target molecules located in the liquid phase. The characteristics of chip hybridization are that the amount of probe is significantly larger than the target gene fragment, and the hybridization kinetics is linear. The strength of hybridization signal was positively correlated with the amount of target gene in the sample.
3. Detection and analysis
After hybridization and cleaning of the chip, the unhybridized molecules are removed, and the target DNA (hybridization molecule) with fluorescent labeling forms a hybrid with its complementary DNA probe. Under laser excitation, fluorescein emits fluorescence. The fluorescence signal is detected and analyzed by scanner, and the information of target DNA is reflected by the original sequence of DNA probe on display.