| Gene chip has been widely applied to all kinds of biological science such as biological diagnosis, biological expression, biological group, disease diagnosis, drug screening, discovery of new genes and the diagnosis of various pathogens, penetrated into all fields of life sciences, and become a powerful tool for the research post-genomic era. In recent years, some gene-chip synthesis platform were yielded by Illumina, Affymetrix, Agilent, Roche-Nimble Gen and others foreign company, including related eagents and kits, database, microarray analysis software tools, chip preparation series platform equipment and spare parts, scanning instrumentation and hybridization equipment. Moreover, the platform was difficultly applied to most of the molecular biology laboratory in our country because of the price of platform more than several million RMB, gene-chip synthesis technology protecting by patents and the high price of synthetic gene chip. So, Research and development of a proprietary gene-chip synthesis platform should be important, which was fast, accurate and low cost platform.This research was developed a low-cost, reliable, simple process, particularly suitable for industrial synthesis of the gene chip platform which used in-situ synthesis of biochip basing on the principle of typography and synthesize methylation-specific oligonucleotide microarray to detect lung cancer. The major content was as follows:1. The synthesizer for preparing gene chip of typography techniqueThe DNA synthesizer was designed and manufactured according to preparing gene chip of typography technique. The template, the reaction tank base, two-dimensional positioning system and the tolerance of assemble between the template and the reaction tank was detected the accuracy by using three-coordinated measurement instrument. The accuracy of printing template and the reaction tank base were 3μn, the position tolerance of assembly between the template and the reaction tank was less than 8μm, X axis repeatability was 8.8μm, X-axis positioning accuracy was 14.4μm, Y axis repeatability was 9μm, y-axis positioning accuracy was 15μm.The testing results showed that the accuracy of the machine was good enough to meet the demand of in-situ synthesis of biochip based on the principle of typography printing.2. Building the microarray platform for typography techniqueThe microarray platform included glove box without anhydrous and oxygen, hybridization instrument, fluorescence scanner and DNA synthesis equipment as to core. The synthesis efficiency of gene chip synthesis platform was affected on the time of imprinting, the volume of the reactor base monomer and the gas pressure of manipulator with imprint. And the completely orthogonal regression experiment was adopted to research the synthesis efficiency. The results showed that the synthetic efficiency of system was 100 percent when imprinted time was 45 second, the volume of the reactor base monomer was 1200 ul and the gas pressure of manipulator was 0.12 MP.Moreover, the oligonucleotide microarray of the fourth exon mutation in p53 gene was produced. The hybridization results showed that the fluorescence intensity is uniform in same oligonucleotide probe of microarray and oligonucleotide microarray could detect single base mismatch and match.3. Methylation-specific PCR method identifying the result of the methylation specific oligonucleotide microarrayMethylation-specific oligonucleotide microarray of RARB, FHIT and RASSF1A gene was synthesized by the gene chip synthesis platform, further, the Methylation-specific oligonucleotide microarray was detected between lung cancer and normal groups. The results of methylation-specific PCR were verified in that of methylation chip. The results indicated there were no association between methylation-specific PCR and methylation-specific oligonucleotide microarray (p>0.05). Methylation specific oligonucleotide chip was a low-cost, reliable, high sensitivity and high specificity of gene chips. And which could be used in clinical research. The in-situ synthesis microarray platform was a cheap, reliable and simple process, particularly suitable for industrial preparation of the gene chip synthesis platform.4. Analysis of lung cancer susceptibility by methylation-specific oligonucleotide microarray Methylation-specific oligonucleotide microarray of RARB, FHIT and RASSF1A gene was synthesized by the gene chip synthesis platform, detecting the lung cancer susceptibility between lung cancer patients and normal people. The results showed that the promoter methylation of RARB and RASSF1A gene was high level and the promoter methylation of FHIT gene was low level.The promoter methylation of FHIT, RARB and RASSF1A gene were candidate molecular markers for early diagnosis of lung cancer.
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