Studies On Slides Substrate Surface Chemistry And Nucleic Acid Fragments Immobilization Of DNA Microarray

DNA microarrays technologies have become one of the key methods for genomic studies. DNA microarrays have been utilized for genetic mutational scanning and polymorphisms analysis, disease screening, antisense reagents selecting, and gene expression monitoring.In the process fabricating DNA microarray, one of the most important process is to immobilize or directly synthesize oligonucleotides on solid supports, such as glass slides. In DNA immobilization process, the glass slide need to be modified so that DNA probes can be immobilized to the slide surface fast, stably and precisely. The immobilization of nucleic acid fragments on the modified glass slide surface is key step in DNA microarray fabrication. Therefore, the research and development in this area will have great implications for the promotion of microarray technology.The immobilization technique of DNA is the cross-subject of surface chemistry and molecular biology. There are two mainly categories for immobilizing oligonucleotides: physical adsorption and covalent linkage. DNA andOligonucleotides immobilized by the former method are unstable and liable to be removed from the surface. Hence, oligonucleotide immobilization by physical adsorption is gradually being substituted by covalent linkage. In this research, we introduced the method of covalent linkage in the glass surfaces modification and DNA immobilization process.Traditional poly-L-lysine coating methods were modified to enhance the immobilization and hybridization efficiency. In this study, we introduced two new approaches of DNA immobilization and surfaces treatment. For one approach is to modify the surface by poly ammoniates, another is to coat the surface with acrylic acid and acrylamide. The slides based on above-mentioned surfaces treatment were validated in X, phage DNA microarrays and SARS coronavirus oligonucleotide microarrays. To detect the SNPs of p53 gene, one of highly mutated genes, the method of an arrayed primer extension based on oligonucleotide microarray was developed.The study could be divided into the following sections:1. Modification of the conventional poly-L-lysine coating protocol. Slides were prepared by covalently coupling of poly-L-lysine to glycidoxy-modified glass surface, and activated by PDITC (1,4-phenylen diisothiocyanate). Oligonucleotides were attached to the activated surface covalently by deposition technology. Some assays were developed, such as the immobilization efficiency and hybridization experiments, which were used to compare the qualities of the modified slides with conventional slides. Results showed that the improved surface exhibited higher immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics, which indicated that the modified slides were superior to those slides treated with conventional approach. And the detection sensitivity of micoarrays was improved greatly.2. Three dimension modification of the substrate surface: Due to the planarsurface structure of microslides, the loading capacity of microarrays is limited. In this paper, we studied two amine-modification procedures for DNA microarray preparation based on covalent linkage method. At the same time, dendrimeric structure and polymers coating were formed on glass surface. The one of proposed approaches was that the silanized slides were coated with poly ammoniates and activated by PDITC (1,4-phenylen diisothiocyanate). This surface was called poly ammoniates system(abbreviated Poly-Amine slides). In the other method, acrylic acid -co-acrylamide copolymer and EDC/NHS (l-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide) were used as coating agents and activator respectively. This surface was called acrylic acid -co-acrylamide copolymer system (abbreviated AACA-copolymer slides). The two modified slides were used in the preparation oligonucletide microarrays. Certain assays were developed, such as the immobilization efficiency, hybridization efficiency and period of validity to review the qualities of the two modified slides. Through the immobilization efficiency experiments, we reached the best immobilization concentration and length of unmodified oligonucletide probe. Hybridization experiments were showed that two surfaces have high fluorescence intensity, low background. The uniform, steady and regular shaped signal spots of the microarrays indicated that the modified surfaces were satisfactory in immobilizing unmodified oligonucleotides. The two platforms were suitable for microarray preparation, and acrylic acid-co-acrylamide copolymer modification was more preferred.3. Comparatively study using microarrays. Surface modified slides based on the above methods were used in the preparation and detection of DNA microarrays and oligonucleotide microarrays in order to observe the general ability. 1) The Poly-Amine and AACA- copolymer slides were used in the preparation of X. phage DNA microarrays. We adopted the technique of PCR for massive amplification of microarray probes. The whole k phage genomic DNA microarray were micro-grided with Cartesian Microarrayer.The DNA samples derived from the 1 phage genome were labeled by restriction labeling method and applied to the microarray for hybridization. The hybridization results were scanned by a confocal scanner. 2) 60 mer SARS-CoV oligonucleotide probes were designed by bioinformatics methods. Twelve oligonucleotides were originally selected, synthesized, and printed onto an oligo microarray for SARS-CoV detection. The practical application indicated that there were perfect immobilization efficiency and detection sensitivity on the modified Poly-Amine and AACA- copolymer platforms. The uniform, steady and regular shaped signal spots of the microarrays indicated that the microarrays were successfully manufactured in comparison with the commercial CMT-GAPS and Super-Amine slides.4. SNP microarray of p53 of primer extension: Increasing evidences imply that SNPs are involved in etiopathology. therapeutical variant and prognosis of many diseases. To detect the SNPs of p53 gene, the method of an arrayed primer extension based on oligonucleotide microarray was developed. Twelve 34 mer specific oligos extension primers were designed in exton 3 of the p53 gene. These primers were synthesized and printed onto the best platform of AACA- copolymer slides. The fragments of p53 DNA were extracted from K562 cells by nested PCR, which applied to the oligo microarray for hybridization. After hybridization, the primer extension reactions were carried out with DNA polymerase (Klenow Fragment), together with Cy3-dUTP or Cy5-dCTP. The fluorescent intensity analysis was evaluated after the washing and scanning steps, and the fragments of p53 were sequenced by ABI3730 DNA analyzer. The conditions of the primer extension reactions were optimized. The scanning result showed that the primer extension reactions were successful, and single base labeled with Cy3 or Cy5 was extended correctly at the end of 3'primers. The results of microarray SNPs detection were consistent with the results of sequencing verification. These results indicated that the arrayed primer extension techniques were useful in parallel detecting SNPs of interest genes, which was not only sensitive and accurate, but also miniaturized the assays whileanalyzing multiple DNA targets with minimal reagents.In summary, to immobilize DNA or oligonucleotide efficiently on modified surface is one of the key technologies in DNA microarray fabrication, which is a prerequisite for the fabrication, and utilization of microarrays in large scale. In this paper, we systematically investigate the methods of covalent linkage in the modification of microarrays surfaces and DNA immobilization process, and developed several novel procedures for DNA microarray platform preparation. The platforms were used in the fabrication and detection of X DNA microarrays, SARS-CoV oligonucleotide microarrays. At the same time, the modified surfaces were further used to detect the SNPs of p53 gene in parallel. The results of the experiments demonstrated that the properties of our novel slides were superior to the commercial ones. Our reported process of the surface preparation is simple, in-noxious and low cost. The contribution of our work may play important roles for laboratory made microarray as well as for development of microarray industry.