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Biochip technology was developed with the progress of the "Human Genome Project (HGP)". It is one of the most far-reaching major scientific and technological developments since the mid-1990s. It integrates A highly interdisciplinary new technology integrating microelectronics, biology, physics, chemistry, and computer science has great basic research value and obvious industrialization prospects. Biochip technology includes gene chips, protein chips, cell chips, tissue chips, and new biochips such as component microarray chips, channel microarray chips, and biosensor chips (1). This article mainly discusses gene chip technology, which provides a powerful tool for the study of gene functions in the "post-genome project" period and will make major breakthroughs in genetic diagnosis, drug screening, and personalized drug delivery. This technology has been rated as One of the world's top ten scientific and technological developments in 1998.

1 Basic concept

Gene chip is also called DNA chip, DNA microarray, and oligonucleotide array. It refers to the use of original In situ synthesis or micro-printing means, tens of thousands of DNA probes are solidified on the surface of the support to produce a two-dimensional DNA probe array, which is then hybridized with the labeled sample and detected by detecting the hybridization signal. To achieve rapid, parallel, and efficient detection or medical diagnosis of biological samples, because silicon chips are commonly used as solid supports and computer chip preparation technology is used in the preparation process, it is called gene chip technology.

2 Basic technical process

2.1 Construction of DNA square array

Select silicon wafer, glass wafer, porcelain wafer or polypropylene film, nylon film, etc. The support is processed accordingly, and then oligonucleotide probes can be synthesized on surfaces such as silicon wafers using photoconductive chemical synthesis and photolithography technology; (2) Either oligonucleotide chain probes are synthesized through liquid phase chemistry, or PCR technology amplifies the gene sequence, then purifies and analyzes it quantitatively. The array replicator (arraying and replicating device ARD), or array machine (arrayer) and computer-controlled robot accurately and quickly quantitatively spot different probe samples on The DNA microarray or chip is obtained by placing the positively charged nylon membrane or silicon wafer at the corresponding position and then cross-linking and fixing it with ultraviolet light (3).

2.2 Preparation of sample DNA or mRNA.

DNA/mRNA samples obtained from blood or living tissue must be amplified to improve reading sensitivity before being labeled as probes. Mosaic Technologies has developed a solid-phase PCR system that is better than traditional PCR technology. They design a pair of bidirectional primers on the target DNA and arrange them on an acrylamide film. This method has no cross-contamination and eliminates the need for liquid phase processing. To avoid the complexity, Lynx Therapeutics proposed another innovative method, namely massively parallel solid-phase cloning. This method can clone tens of thousands of DNA fragments in a sample at the same time without separation. and processing each clone individually to make sample amplification more efficient and faster (4).

During the PCR amplification process, samples must be labeled at the same time. Labeling methods include fluorescent labeling, biotin labeling, isotope labeling, etc.

2.3 Molecular hybridization

Complementary hybridization of sample DNA and probe DNA should select and optimize hybridization conditions according to the type and length of the probe and the application of the chip. If used for gene expression monitoring, hybridization requires low stringency, low temperature, long time, and high salt concentration; if used for mutation detection, the hybridization conditions are opposite (5). The characteristics of chip molecular hybridization are that the probe is solidified and the sample is fluorescently labeled. A large number of biological samples can be detected and analyzed at one time, and the hybridization process only takes 30 minutes. The American company Nangon uses a method of controlling electric fields to reduce the molecular hybridization speed to 1 minute or even a few seconds (6). Jorg Hoheisel from the German Cancer Institute and others believe that using peptide nucleic acid (PNA) as a probe is more effective.

2.4 Detection and analysis of hybridization patterns

Use laser to excite the sample on the chip to emit fluorescence. Strictly paired hybrid molecules have higher thermodynamic stability and strong fluorescence; Completely hybridized double-bonded molecules have low thermodynamic stability and weak fluorescence signal (less than 1/35~1/5 of the former) (2), while non-hybridized molecules have no fluorescence. The signals at different sites are detected by laser focus microscopy or epifluorescence microscopy, and are processed and analyzed by computer software to obtain the relevant gene map. At present, methods such as mass spectrometry, chemiluminescence, and optical fiber methods are more sensitive and faster, and have a tendency to replace fluorescence methods.

3 Applications

3.1 Sequencing

Gene chips use the hybridization pattern generated by molecular hybridization of fixed probes and samples to arrange the sequence of the sample to be tested. , this determination method is fast and has very attractive prospects. Mark chee et al. used an array containing 135,000 oligonucleotide probes to determine the full-length human mitochondrial genome sequence of 16.6 kb, with an accuracy of 99% (7). Hacia et al. used a high-density microarray containing 48,000 oligonucleotides to analyze the sequence differences between chimpanzee and human BRCA1 genes. The results found that the nucleic acid sequence homology in the approximately 3.4 kb length of exon 11 ranged from 98.2% to 83.5%. time, suggesting a high degree of evolutionary similarity between the two (8).

3.2 Detection of gene expression levels.

Expression level detection using gene chips can automatically and quickly detect the expression of thousands of genes. Schena et al. used a cDNA microarray of up to 45 genes in the Arabidopsis genome (14 of which are complete sequences and 31 are ESTs) to detect the expression levels of these genes in the root and leaf tissues of the plant, using different colors. The fluorescein-labeled reverse transcription products were hybridized to the microarray respectively. After laser focus microscopy scanning, it was found that there were expression differences in 26 genes in the root and leaf tissues of the plant, and the CAB1 gene involved in chlorophyll synthesis was expressed differently in the leaves. The tissue expression is 500 times higher than that of root tissue. (9) Schena et al. used a cDNA library of human peripheral blood lymphocytes to construct a cDNA microarray representing 1046 genes to detect the differences in gene expression of T cells cultured in vitro in response to heat shock, and found that 5 genes were significantly altered after treatment. There was very obvious high expression, with 11 genes having moderately increased expression and 6 genes having significantly suppressed expression. This result was also confirmed using fluorescein exchange labeling of control and treatment groups and Northern blotting (10). After the completion of HGP, genome chips for detecting expression changes of all human genes under different physiological and pathological conditions are not far away (11).

3.3 Gene diagnosis

A standard pattern can be obtained by isolating DNA from the genome of normal people and hybridizing it with a DNA chip. The lesion map can be obtained by isolating DNA from the patient's genome and hybridizing it with a DNA chip. By comparing and analyzing these two maps, the DNA information of the lesion can be obtained. This gene chip diagnostic technology will become a new modern diagnostic technology with its characteristics of rapidness, efficiency, sensitivity, economy, parallelization and automation. For example, Affymetrix integrates the full-length sequence of the P53 gene and probes of known mutations on a chip to form a P53 gene chip, which will play a role in early diagnosis of cancer. For another example, Heller et al. constructed a 96-gene cDNA microarray for detection and analysis of genes related to rheumatoid arthritis (RA) to explore the application of DNA chips in the diagnosis of infectious diseases (12). Now, a series of diagnostic chips such as hepatitis virus detection diagnostic chips, Mycobacterium tuberculosis drug resistance detection chips, and various malignant tumor-related virus gene chips are gradually entering the market. Genetic diagnosis is the most commercially valuable application of gene chips.

3.4 Drug Screening

How to isolate and identify the active ingredients of drugs is a major obstacle encountered by the traditional Chinese medicine industry and the development of traditional Western medicine. Gene chip technology can solve this obstacle. It is an effective method that can be used for large-scale screening, has strong versatility, and can explain the mechanism of action of drugs from the genetic level. That is, gene chips can be used to analyze the differences in gene expression in different tissues and organs of the body before and after medication. If you then use mRNA to construct a cDNA expression library, and then use the obtained peptide library to make a peptide chip, you can screen out some of the active substances from numerous drug ingredients.

Alternatively, RNA and single-stranded DNA are very flexible and can form complex spatial structures, which are more conducive to combining with target molecules. The RNA or single-stranded DNA in the nucleic acid library can be fixed on the chip and then incubated with the target protein. , forming protein-RNA or protein-DNA complexes, which can screen specific drug proteins or nucleic acids, so the combination of chip technology and RNA libraries will be widely used in drug screening. In the search for HIV drugs, Jellis et al. used combinatorial chemical synthesis and DNA chip technology to screen 654,536 phosphorothioate octameric nucleotides and identified inhibitors with XXG4XX-like structures. Experiments have shown that this screened substance is effective against HIV. There is a significant blocking effect on infected cells. (13) Biochip technology greatly increases the speed of drug screening, target gene identification and new drug testing, and greatly reduces the cost. Gene chip drug screening technology has just started. Many pharmaceutical companies in the United States have begun preliminary work, that is, they are establishing expression profile databases to provide various target genes and analysis methods for drug screening. This technology has great potential application value.

3.5 Personalized administration

Clinically, the same dose of drug that is effective for patient A may not be effective for patient B, and may have side effects for patient C. Patient responses vary widely in terms of drug efficacy and side effects. This is mainly due to differences in patient genetics, such as drug response genes, which lead to different responses to drugs. For example, the cytochrome P450 enzyme is related to the metabolism of about 25% of widely used drugs. If a patient's gene for this enzyme is mutated, it will have significant side effects on the antihypertensive drug isoquine. About 5% to 10% of Caucasians are deficient in this enzyme. enzyme gene activity. It is now clear that there are widespread variations in these genes, which, in addition to producing different responses to drugs, are also associated with susceptibility to various diseases such as tumors, autoimmune diseases, and Parkinson's disease. If gene chip technology is used to diagnose patients first and then issue prescriptions, individual optimized treatment can be implemented for patients. On the other hand, in treatment, the specific causes of many of the same diseases vary from person to person, and the medications should also vary from person to person. For example, there are many subtypes of hepatitis B, and multiple sites in the HBV gene such as the S, P and C gene regions are prone to mutations. If the hepatitis B virus gene polymorphism detection chip is used to detect hepatitis B virus at regular intervals, it is very meaningful to guide medication to prevent hepatitis B virus drug resistance. For another example, the drugs currently used to treat AIDS are mainly inhibitors of viral reverse transcriptase RT and protease PRO. However, drug resistance often occurs after 3 to 12 months of medication. The reason is that one or more spots are generated in the rt and pro genes. mutation. The four common mutation sites of the Rt gene are Asp67→Asn, Lys70→Arg, Thr215→Phe, Tyr and Lys219→Glu. Mutations at all four sites increase the drug tolerance hundreds of times compared with a single site mutation (14 ). If the entire sequence of these gene mutation sites is constructed as a DNA chip, it can quickly detect whether the patient has mutations in one or that gene or multiple genes, and can prescribe the right medicine, so it is of great significance to guide treatment and prognosis.

In addition, gene chips have great application value in new gene discovery, pharmacogenomic mapping, identification of traditional Chinese medicine species, and DNA computer research.

4 Current status and prospects of gene chips at home and abroad

Since 1996, the American company Affymetrix successfully produced the world's first biochips for drug screening and laboratory testing, and A chip system was produced (15). Since then, countries around the world have made rapid progress in chip research and continued to make new breakthroughs. Hyseq, Syntexi, Nanogen, Incyte in the United States and various countries in Japan and Europe are actively carrying out DNA chip research; multinational companies such as Motorola, HP, and IBM have also invested heavily in chip research. In December 1998, Affymefrix and Molecular Dynamics announced the establishment of the Genetic Analysis Technology Consortium to develop a unified technology platform to produce more effective and affordable equipment. In response, the British Amershcem Pharmacia Biotechnology also On the same day it was announced that the partially mastered technology would be made available to promote the application of this technology (16).

The United States held two conferences on chip technology, at which President Clinton highly praised and affirmed the technology and regarded gene chips as a compass to ensure lifelong health (17). It is expected that the sales of biochips will reach 20-30 billion US dollars in the next five years; according to Fortune magazine's prediction (97.3), in the 21st century, the impact of biochips on human beings will likely exceed that of microelectronic chips.

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