| Aquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) has aroused wider and wider attentions because it can lead to severe damage. As a result, early diagnosis is urgently needed. An ideal diagnosis method is one that can detect and genotype the HIV gene at the same time, and can monitor the coexisted infection during the development of the AIDS. The recently developed DNA chip technology is a powerful method that provides researchers with the opportunity not only to detect the AIDS at early stage in a more sensitive, specific, speedy, efficient and automatic manner, but also to detect and genotype HIV gene simultaneously.DNA chip technology involves several experiment procedures including array manufacturing either by in situ synthesis of oligonucleotide probes or by spoting a large quantity of DNA probes directly onto the surface of the substrate, hybridization of the chip with labeled samples, detection and analysis of the hybridization signals, and aquiring of the gene sequencing and gene expression profiling information. DNA chips can be classified into two categories according to their preparations: in situ synthesis genechip and DNA microarray, of which, DNA microarray has become more and more popular among many research institutes and developed very quickly because of less patent protection. The key points of the DNA microarray technology include probe preparation, sample labeling and hybridization. In this experiment, the restriction display (RD) technique invented by Wenli Ma and Wenling Zheng was not only applied to the isolation of large quantity of gene fragments in order with high efficiency for DNA chip manufacturing, but also wisely used in sample labeling and hybridization process which led to dramatic improvement in hybridization efficiency.To begin with, RD method was applied to prepare for the restriction display probes of HIV-1 gene. The HIV genes were digested with Sau3A I in order to isolate restriction fragments with suitable length, and the adapteswere linked to both ends of the fragments, then the universal primers were designed according to the sequence of the enzyme restriction sites and the adapters, one or two bases were elongated at the 3' end of the universal primers to design four or sixteen selective primers, with which combination PCR reactions were divided into ten or several subgroups with primer combinations. PCR products from each subgroup were purified and cloned into T vectors for sequencing analyses, and then the second or third round PCR was performed using recombinant plasmids, directly purified PCR products or retracted PCR products from agarose gel as templates, about tea or more HIV gene fragments ranged from lOObp to 1000 bp were isolated from each subtype.The gene fragments amplified above were various in their length, stucture, sequence and charictaristics etc., so hybridization analysis should be performed to screen the probes suitable for detection. In this experiment, Cartesian PixSys 5500 arrayer was used to spot the RD fragments onto the aminosilane coated slides to manufacture the gene chip for each subtype, and the HIV samples were labeled with fluorescence by random primering method and hybridized with chip, then the slides were washed, dried and scanned using ScanArray Lite, and the hybridization signal intensities were analyzed. The DNA microarray technology was studied systematically, so the optimate probes were selected, and the sequence, stucture, G+C content and Tm value etc. were analyzed. Then the selected probes from each subtype were arrayed onto a single chip in order, and hybridized with each subtype for HIV genotyping analysis. The results showed that these RD fragments from each subtype on the chips could hybridize specifically to the fluorescently labeled HIV DNA samples, and specific hybridization patterns for each sutype were shown also. It demonstrates that RD technology is a quick, easy-to-use, practical method for gene chip probe preparation.The RD technology was also applied to sample...
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