Studies Of Novel Letterpress Printing Method And Substrates For The In Situ Synthesis Of DNA Microarrays

DNA microarrays have emerged as a powerful tool in numerous demonic applications such as gene expression and genotyping applied towards disease understanding and drug development. At present there are two major trends of development on DNA chips, the one is toward gene chips with high automation, miniaturization and high throughput screening, Affymetrix Corporation is the only producer that achieved DNA probe arrays with high spatial resolution by using photolithography and solid-phase DNA synthesis. The other is towards middle, low-density chips which meet widely applications of actually clinical diagnosis, military affairs and judicature. With the completion of the Human Genome Project and further development of Functional Genome Research, tremendous biological analyses of many genes demand further improvements of reliable, quantitative, highly sensitive and highly specific detection methods. These have ushered in challenges and opportunities for advance of DNA chip technology.However, the fabrication of such miniaturized devices faces several challenges, and the major among those are cost,synthetic efficiency and quality. For example, the conventional DNA chips are priced around from $150,000(US$1,220) to $350,000, which is too high to accept in market. While the Affymetrix's method remains to be improved appropriately to address some limitations such as high cost and lower synthetic efficiency. According to current status of gene-chip development, we developed a method to synthesize oligonucleotide arrays in situ on glass and polypropylene surfaces by using the Letterpress Printing Method, which has the highspatial resolution, lower cost, reliable operation, and high synthetic efficiency. This dissertation describes works involved in developing a method to synthesize oligonucleotide arrays in situ on glass and polypropylene surfaces and two gene-chip substrates in accordance with mentioned above. The major contributions are as follows:1. The dissertation presents an approach of preparing substrate for in situ synthesis of oligonucleotideon microporous polyamide-6 membranes substrate after its hydrolysis, which required the membranes immersion into a medium of a 0.01 mol/L NaOH/(H2O-CH3OH) mixture with about 36 hours refluxing. The emergence of amines (NH2) on the membranes surface was demonstrated by Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) characterization. Optimum hydrolyzing conditions were determined through the ultra-violet (UV) spectra. A pH value of 12 and a hydrolysis time of 36 h are the best conditions for the modification. The treated membrane can be applied to in situ synthesis of oligonucleotide and, for example, the oligonucleotide probes of 5'-AAC CAC CAA ACA CAC-3'were successfully synthesized on the modified membranes. The single step coupling efficiency is above 98 % determined by ultraviolet (UV) spectra.2. The second substrate for in situ synthesis of oligonucleotide has been prepared by using low-pressure plasma technique. Polypropylene microporous membrane and slice were treated with plasma in a mixture of nitrogen and hydrogen (a ratio of 1:2 in volume). The success of amino functional groups grafting was demonstrated by characterizations of Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) , X-ray photoelectron spectroscopy (XPS) and ultra-violet (UV) spectra. The density of polar amino groups on the membrane surface was approximately 0.5μmol/cm2, which is much higher than that of amino functional groups on modified glass slides. The amino-treated membranes were successively applied to the in situ synthesis of oligonucleotides and an averagecoupling yield of more than 98% on the substrate was achieved. These modified microporous membranes open prospects for the in situ synthesis of DNA microarrays.3. The dissertation also compared results of oligonucleotide probes arrays in-situ synthesized on three substrates, which were different plasma-functionalzed PP slice surfaces of the hydrogen and nitrogen plasma modified Polypropylene (PP) slice surface, those the amino plasma modified PP slice surface, and oxygen plasma and silanazing modified PP slice surface, respectively. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of polar amino groups on the plasma and silanazing treated PP slice surfaces. The specificity of the probe arrays was discriminated by hybridizing with fluorescent target sequences. The positive results indicated that all the three substrates could serve as good substrates for high-density oligonucleotides arrays synthesis and DNA chip fabrication. Among them, the hydrogen and nitrogen plasma-functionalzed PP slice surface was the best one .4. Oligonucleotide probe arrays were in-situ synthesized on the H2/N2 plasma modified poly(terafluoroethylene) (PTFE) surface via micro-fluidic channels connected with an automated DNA synthesizer. A contact angle measurement of water droplets was used to ascertain the hydrophilicity of the modified surface. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of polar amino groups on the surface. Ultra-violet (UV) spectrum analysis indicated that the surface showed a coupling efficiency higher than 98% for in situ synthesis of oligonucleotides arrays. The probe array specificity was discriminated by hybridizing with fluorescent target sequences. Oligonucleotide probe arrays on modified PTFE surface showed high stability and durability after repetitive denaturing and hybridizing. The results implied the plasma modified PTFE surface was extremely stable, excellent performance in DNA hybridization assays and could service as a good substrate for high-density oligonucleotide array synthesis.5. The dissertation developed a method to synthesize oligonucleotide arrays in situ on glass and polypropylene surfaces by using the Letterpress Printing Method,which has the high spatial resolution, lower cost, reliable operation, and high synthetic efficiency. The principle and process of the Letterpress Printing Method for fabricating DNA chip has been described in detail. The conditions of oligonucleotide array synthesis were optimized in the dissertation. Such as, the influence of coupling reaction on reagent characters, the effect of coupling efficiency on block reagent, the activity of mixture solution for tetrazole and oligonucleotide monomer, and so on, mentioned above were particularly discussed in the dissertation. The oligonucleotide arrays of 16 and 160 size were synthesized with four different sequences probes by using above option conditions. Four specific oligonucleotide probes including the matched and the mismatched by the target sequence gave obviously different hybridization fluorescent signals, which showed that the genechip fabricated with the Letterpress Printing method can be used to rapidly screen single-nucleotide polymorphisms.