Experimental procedure
1. Chip preparation
There are two basic methods for the preparation of gene chips, one is the on-chip synthesis method, and the other is the spotting method. The on-chip synthesis method is based on the principle of combinatorial chemistry. It uses a set of positioning templates to determine the coupling sites and order of different chemical monomers on the substrate surface. The key to the preparation of DNA chips in on-chip synthesis is the intricate combination of template positioning technology with high spatial resolution and solid-phase synthesis chemical technology. At present, there are a variety of template technologies for on-chip synthesis of gene chips, such as photodeprotection parallel synthesis method, photoresist protection synthesis method, microfluidic template solid phase synthesis technology, molecular stamping multiple in-situ synthesis Method, spray printing synthesis method. The on-chip synthesis method can take advantage of microfabrication technology, and is very suitable for making large-scale DNA probe array chips to realize the standardization and large-scale production of high-density chips.
Gene chip spotting method first prepares the cDNA (or oligonucleotide) probe library according to the conventional method, and then distributes different probe solutions through glass, nylon or other solid phases by special needles and micro-spraying heads Different positions on the surface of the substrate, and the probe is fixed to the corresponding positions of the chip through the combination of physical and chemical. This approach is more flexible. Probe fragments can come from multiple sources. In addition to oligonucleotide probes, longer gene fragments and nucleic acid analog probes (such as PNA, etc.) can also be used. Probe preparation methods can use conventional DNA probe synthesis methods, or PCR amplified cDNA, EST libraries, etc. There are many ways to fix it. The advantage of the spotting method is that it can make full use of the original oligonucleotide synthesis method and instrument or cDNA probe library, the length of the probe can be arbitrarily selected, and the fixing method is relatively mature, and the flexibility is suitable for the research unit According to the need to prepare scientific research-type gene chips, make small-scale commercial gene chips.
2. Preparation of target gene samples
As with ordinary molecular biology experiments, the preparation of target genes requires the use of conventional means to extract template molecules from cells and tissues for template amplification and labeling. The gene chip includes a large number of probe molecules. Therefore, the preparation method of the target gene sample will be determined according to the type of the gene chip and the research object (such as mRNA, DNA, etc.). For most genes, the expression level of mRNA roughly corresponds to the level of its protein. Therefore, quantitative detection of mRNA expression levels in cells is important for understanding the nature and state of cells. The gene chip can be used to detect the mRNA expression differences of a large number of genes in the cell. The preparation of the target gene is generally performed by RT-PCR using oligo dT as a primer for amplification.
The label of the sample to be tested mainly uses fluorescent molecules. The conventional labeling process is to add fluorescently labeled dNTPs (at least one of which is fluorescently labeled) during the amplification process. Fluorescently labeled single nucleotide molecules are introduced into newly synthesized DNA fragments during transcription and replication. . After the latter is denatured, it can be molecularly hybridized with the microprobe array on the gene chip. The end labeling method can also be used to directly label fluorescence on the primer. That is, fluorescent primers are prepared by applying fluorescently labeled dNTPs during primer synthesis, or by biotin labeling for fluorescent labeling.
For gene chips with a low array density (often using membranes as substrates), the isotope detection method can be used, and 32P labeling technology can be used, so that the commonly used isotope imaging technology and instruments can now be used. However, in the process of using the isotope target gene labeling method, some lattices with high expression levels and strong hybridization signals are likely to produce halos around them. When the hybridization signals of the surrounding lattices are weak, their hybridization signals are easily subject to strong hybridization Masking of the signal.
3. Hybridization of target genes and detection of their signals
The hybridization process of the cDNA gene chip and the target gene is basically the same as the general conventional molecular hybridization process. In the hybridization detection of gene chips, in order to better compare the gene expression differences of samples from different sources, or to improve the accuracy and measurement range of gene chip detection, multi-color fluorescence technology is usually used. That is, target genes of different sources are modified with fluorescent probes of different excitation wavelengths, and at the same time, they are hybridized with the gene chip. By comparing the distribution of fluorescence at different wavelengths on the chip, the differences in gene expression in different samples can be directly obtained.
People have also developed other gene chip hybridization detection methods with high sensitivity and good specificity. For example, short sequence coupling method. This method first completely hybridizes the non-labeled DNA target sequence with the probe array on the gene chip. If the fixed end of the probe on the gene chip is the 5 'end, continue to use the target gene as a template, which can be exposed to the solution On 3'-OH, new fluorescently labeled bases (ddNTPs) were synthesized with DNA polymerase. The gene of the target sequence can be distinguished by detecting ddNTP.
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