Silica Based Specific Adsorbents for DNA Purification

Abstract

The aim of this study is to prepare silica based affinity sorbents for the purification of plasmid DNA and ge- nomic DNA. For this purpose, 3-aminopropyl trimethoxysilane APTS is chosen for surface modification of silica particles average size: 100-150 μm and silica based affinity sorbent carrying NH2 groups on its surface is obtained. APTS modified and unmodified silica particles are characterized by elemental analysis. Adsorption of genomic DNA on the APTS modified silica particles is performed in continuous and batch systems. Effect of io- nic strength, pH, temperature, initial genomic DNA concentration and flow rate is investigated. In the last part of the study, pEGFP-N3 plasmid DNA is separated from E.coli cells by using the APTS modified silica particles and the results are compared with commercial Qiagen DNA purification kit. The results may be summarized as follows: amount of NH2 groups on the APTS modified silica particles is in the range of 38-60 mmol/g. Maximum adsorption capacity of the APTS modified silica particles is found to be 49.3 mg/g and this value is obtained at pH 7.0. Genomic DNA adsorption capacity of the APTS modified silica particles is increased in continuous system. It is also shown that the APTS modified silica particles can be used after ten adsorption-desorption cycle without any significant decrease in adsorption capacity. The APTS modified silica particles are used for adsorption of plasmid DNA from E.coli cells and adsorption capacity is found to be 0.21 mg/g in continuous system.

Keywords

• Silica,plasmid DNA,DNA separation,pEGFP-N3

DNA Saflaştırılması İçin Silika Temelli Taşıyıcılar

Abstract

B u çalışmanın amacı, plazmid DNA ve genomik DNA’nın ayrılması için silika temelli afinite sorbentlerin hazırlanmasıdır. Bu amaçla, silika partiküllerin ortalama boyut: 100-150 µm yüzeyinin modifikasyonu için 3-aminopropiltrimetoksisilan APTS seçilmiş ve yüzeyinde NH2 grubu taşıyan silika temelli afinite sorbent elde edilmiştir. APTS ile modifiye edilmiş ve edilmemiş silika partiküller elementel analiz ile karakterize edilmiştir. APTS modifiye silika partiküller üzerine genomik DNA adsorpsiyonu sürekli ve kesikli sistemde gerçekleştirilmiştir. İyonik şiddetin, pH’nın, sıcaklığın, başlangıç genomik DNA derişiminin ve akış hızının etkisi araştırılmıştır. Çalışmanın son aşamasında APTS modifiye silika partiküller kullanılarak, pEGFP-N3 plazmid DNA E.coli hücrelerinden ayrılmıştır ve sonuçlar ticari Qiagen DNA saflaştırma kiti ile karşılaştırılmıştır. Sonuçlar şöyle özetlenebilir: APTS modifiye silika partiküller üzerindeki NH2 gruplarının miktarı 38-60 mmol/g arasındadır. APTS modifiye silika partiküllerin maksimum adsorpsiyon kapasitesi 49.3 mg/g olarak bulunmuştur ve bu değer pH 7.0’de elde edilmiştir. APTS modifiye silika partiküllerin genomik DNA adsorpsiyon kapasitesi sürekli sistemde artmıştır. Adsorpsiyon kapasitesinde önemli bir düşüş olmadan APTS modifiye silika partiküllerin on adsorpsiyon-desorpsiyon döngüsünden sonra tekrar kullanılabileceği gösterilmiştir. APTS modifiye silika partiküller E.coli hücrelerinden plazmid DNA adsorpsiyonu için kullanılmış ve adsorpsiyon kapasitesi sürekli sistemde 0.21 mg/g olarak bulunmuştur

Keywords

• Silika,plazmid DNA,DNA ayırma,pEGFP-N3

References

1. 1. A. Jungbauer, Continuous downstream processing of biopharmaceuticals, Trends Biotechnol., 31 (2013) 479.
2. 2. S.C. Tan, B.C. Yiap, DNA, RNA and protein extraction: the past and the present, J. Biomed. Biotechnol., 2009 (2009) 1.
3. 3. A.T. Hanke, M. Ottens, Purifying biopharmaceuticals: knowledge-based chromatographic process development. Trends Biotechnol., 32 (2014) 210.
4. 4. T. Matosa, J.A. Queiroz, L. Bülow, Plasmid DNA purification using a multimodal chromatography resin, J. Mol. Recognit., 27 (2014) 184.
5. 5. G.N.M. Ferreira, G.A. Monterio, D.M.F. Prazeres, J.M.S. Cabral, Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications, Trends Biotechnol., 18 (2000) 380.
6. 6. G.N.M. Ferreira, Chromatographic approaches in the purification of plasmid DNA for therapy and vaccination, Chem. Eng. Technol., 28 (2005) 1285.
7. 7. M.M. Diogo, J.A. Queiroz, D.M.F. Prazeres, Chromatography of plasmid DNA. J. Chromatogr. A, 1069 (2005) 3.
8. 8. A. Sousa, F. Sousa, J.A. Queiroz, Advances in chromatographic supports for pharmaceutical-grade plasmid DNA purification, J. Sep. Sci., 35 (2012) 3046.

9. 9. X.W. Chen, Q.X. Mao, J.W. Liu, J.H. Wang, Isolation/ separation of plasmid DNA using hemoglobin modified magnetic nanocomposites as solid-phase adsorbent, Talanta, 100 (2012) 107.
10. 10. C.L. Chiang, C.S. Sung, C.Y. Chen, Application of silica–magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells, J. Magn. Magn. Mater., 305 (2006) 483.
11. 11. X. Min, P. Han, H. Yang, H. Kim, M. Tong, Influence of sulfate and phosphate on the deposition of plasmid DNA on silica and alumina-coated surfaces, Colloid Surface B., 118 (2014) 83.
12. 12. K. Smerkova, S. Dostalova, M. Vaculovicova, J. Kynicky, L. Trnkova, M. Kralik, V. Adam, J. Hubalek, I. Provaznik, R. Kizek. Investigation of interaction between magnetic silica particles and lambda phage DNA fragment, J Pharm Biomed Anal., 86 (2013) 65.
13. 13. A. Carré, V. Lacarrière, W. Birch, Molecular interactions between DNA and an aminated glass substrate, J Colloid Interface Sci. 260 (2003) 49.
14. 14. Z. Shan, X. Li, Y. Gao, X. Wang, C. Li, Q. Wu, Application of magnetic hydroxyapatite nanoparticles for solid phase extraction of plasmid DNA, Anal Biochem., 425 (2012) 125.
15. 15. O.Z. Nanassy, P.V. Haydock, M.W. Reed, Capture of genomic DNA on glass microscope slides, Anal Biochem., 365 (2007) 240.
16. 16. J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: Laboratory Manuel, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring, NY, 1989.
17. 17. D. Shao, A. Xia, J. Hu, C. Wang, W. Yu, Monodispersed magnetite/silica composite microspheres: Preparation and application for plasmid DNA purification, Colloids Surf. A, 322 (2008) 61.
18. 18. C. Türkcan, S. Akgöl, A. Denizli, Silanized polymeric nanoparticles for DNA isolation, Mater Sci Eng C Mater Biol Appl., 33 (2013) 4498.
19. 19. I. Perçin, E. Sağlar, H. Yavuz, E. Aksöz, A. Denizli, Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification, Int. J. Biol. Macromol., 48 (2011) 577.
20. 20. I. Perçin, V. Karakoç, S. Akgöl, E. Aksöz, A. Denizli, Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate, Mater Sci Eng C Mater Biol Appl., 32 (2012) 1133.
21. 21. F. Sousa, D.M.F. Prazeres, J.A. Queiroz, Dynamic binding capacity of plasmid DNA in histidine-agarose chromatography, Biomed. Chromatogr., 21 (2007) 993.
22. 22. H.P. Zhang, S. Bai, L. Xu, Y. Sun, Fabrication of monosized magnetic anion exchange beads for plasmid DNA purification, J. Chromatogr. B, 877 (2009) 127.
23. 23. R. Fu, C. Wang, J. Zhuang, W. Yang, Adsorption and desorption of DNA on bovine serum albumin modified gold nanoparticles, Colloids Surf. B, 444 (2014) 326.
24. 24. C.H. Liu, S. L. Sahoo, M.H. Tsao, Acridine orange coated magnetic nanoparticles for nucleus labeling and DNA adsorption, Colloids Surf. B, 115 (2014) 150.
25. 25. F. Smrekar, A. Podgornik, M. Ciringer, S. Kontrec, P. Rasport, A. Strancar, M. Peterka, Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns, Vaccine, 28 (2010) 2039.