Immobilization of DNA on Non-porous Polymer Particles of Microsize and Their Application

• Main Authors: Gow-Yuan Lee, 李國源
• Other Authors: Wen -Chien Lee
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/73578344620786857527

碩士 === 國立中正大學 === 化學工程研究所 === 89 === Immobilized oligonucleotide has been widely used in molecular biology, clinical analysis and other fields. One of the promising applications is column chromatography, that involves a use of immobilized DNA fragments on the solid support and has been widely used for the isolation and analysis of polynucleotides and DNA binding proteins. By constructing columns with a single-stranded (ss) polynucleotide covalently attached to the support, single-stranded polynucleotides which can base pair with the attached sequence will be retained by the column. This technique for sequence-specific separation of polynucleotides is called DNA affinity chromatography. In this study, monomers of glycidyl methacrylate (GMA) and styrene were used in the copolymerization. The resultant non-porous copolymerized beads(2.3 μm) were chemically modified to introduce either primary or secondary amino groups, which were ready for the attachment of polynucleotides. The prepared polynucleotide-immobilized beads were the packed into columns(5 cm ´ 0.46 cm I.D.) for the affinity chromatography of complementary stands of nucleic acids. The immobilized ssDNA could capture the complementary polynucleotide with high affinity, and the complementary polynucleotide was strongly retained in the column due to the DNA-DNA base pairing under the adsorption conditions. The single-strand polynucleotide (anti-primer 1) adsorbed at 0.8 M NaCl could be effectively eluted out from the column at about 7 min using 0.4 N NaOH as the elution buffer. In the column chromatography using particles with immobilized DNA via primary amino groups, the non-specific adsorption of polynucleotides became significant when the NaCl concentration was smaller than 0.8 M in the adsorption buffer. To the contrast, the non-specific adsorption of polynucleotides was insignificant in the column using DNA-immobilized particles vis secondary amino groups. This suggests that non-specific adsorption was due to the electrostatic interaction between the polynucleotide and amino group on the particle surface, and this interaction could be weaken by a high salt concentration. However, the higher salt concentration could result in a portion of anti-primer 1 that did not base pair with the immobilized primer 1 and was eluted as the non-retained component. The affinity columns were also useful for the separation of denatured PCR products. The ssDNA that could not base pair with immobilized primer 1 was eluted from the column as the non-retained component, while the strand that was complementary to the immobilized primer 1 was retained under the adsorption conditions and eluted from the column by following application of 0.4 N NaOH. Finally, the results obtained in this study suggest that the prepared affinity column are very useful and worthy to be further studied.