Affinity chromatography of DNA on nonporous copolymerized particles of styrene and glycidyl methacrylate with immobilized polynucleotide.
Nonporous particles of microsize were prepared by the dispersion polymerization of styrene and glycidyl methacrylate and chemically modified to introduce amino groups on the surface by grafting with either hexamethylenediamine or N-methyl-1,3-propanediamine. Aminated particles were then coupled with phosphorylated single-stranded polynucleotides at the 5'-end through covalent linkages. The affinity columns packed with these prepared polynucleotide-immobilized particles effectively retained single-stranded DNA, which could base-pair with the immobilized sequence. Bound DNAs could be eluted to yield a sharp peak by using an aqueous solution of 0.4M NaOH. The nonspecific adsorption due to the electrostatic interaction between the polynucleotide and the residual amino groups on the particle surface via the amination with hexamethylenediamine was significant and could only be reduced by using a high salt (NaCl) concentration. A higher salt concentration in the elution solution could result in a portion of complementary polynucleotide eluted in the nonretained fraction. However, the nonspecific adsorption of polynucleotides was insignificant in the column packed with DNA-immobilized particles prepared via amination using N-methyl-1,3-propanediamine. The column was effective for microanalysis of sequence-specific DNA.
Lee
Gow-Yuan
GY
Department of Chemical Engineering, National Chung Cheng University, Chiayi 621, Taiwan.
Chen
Chi-Hao
CH
Wang
Tzu-Hsien
TH
Lee
Wen-Chien
WC
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis; D004852:Epoxy Compounds / Q000737:chemistry*; D008689:Methacrylates / Q000737:chemistry*; D008969:Molecular Sequence Data; D011108:Polymers / Q000737:chemistry*; D011119:Polynucleotides / Q000737:chemistry*; D016062:Porosity; D012965:Sodium Chloride; D012972:Sodium Hydroxide; D020058:Styrene / Q000737:chemistry*; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D004852:Epoxy Compounds; D008689:Methacrylates; D011108:Polymers; D011119:Polynucleotides; D020058:Styrene; D012965:Sodium Chloride; D012972:Sodium Hydroxide; D004247:DNA; C007870:glycidyl methacrylate