Research Article
Year 2021,
Volume: 43 Issue: 3, 226 - 231, 30.09.2021
Abstract
References
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- 7. Sentchilo V, Mayer AP, Guy L. et al. Community-wide plasmid gene mobilization and selection. ISME J. 2013; 7: 1173–1186. doi: 10.1038/ismej.2013.13
- 8. Shan Z, Wu Q, Wang X. et al. Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles. Anal Biochem. 2010; 398(1): 120-2. doi: 10.1016/j.ab.2009.11.006.
- 9. Stadler J, Lemmens R, Nyhammar T. Plasmid DNA purification. J Gene Med. 2004; 6 (1): S54-66. doi: 10.1002/jgm.512.
- 10. Stewart EJ. Growing unculturable bacteria. J. Bacteriol. 2012; 194: 4151–4160. doi: 10.1128/JB.00345-12.
- 11. Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology 2009; 1-10. http://dx.doi.org/10.1155/2009/574398
- 12. Tseng YC, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA. Adv Drug Deliv Rev. 2009; 61:721-731. doi: 10.1016/j.addr.2009.03.003
- 13. Warburton PJ, Allan E, Hunter S. et al. Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease, using transposon-aided capture (TRACA). FEMS Microbiol. Ecol. 2011; 78: 349–354. doi: 10.1111/j.1574- 6941.2011.01166. x
- 14. Yeung BZ, Lu Z, Wientjes GM. et al. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA. AAPS J. 2015; 17(6): 1475-82. doi: 10.1208/s12248-015-9812
Year 2021,
Volume: 43 Issue: 3, 226 - 231, 30.09.2021
Abstract
Objective: The use of gene therapy for targeted therapy in various genetic diseases has become more and more popular day by day. Various carrier vectors are used in gene therapy. The plasmid we are interested in in this study is used as a gene delivery vector. In this study, it was aimed to perform the isolation and characterization studies of the peGFP-N1 plasmid. In addition, necessary properties were investigated to make it a suitable gene carrier vector.
Method: In this study, isolation was performed using the plasmid peGFP-N1 via classical method. Escherichia coli was used as expression medium in the isolation process. In order to calculate the amount of plasmid DNA in the studies, the spectral curve value of the plasmid DNA was calculated. Spectrophotometric methods were used to determine the amount and impurity of the isolated bacterial DNA.
Results: In plasmid DNA isolation studies, it was observed that the yield was quite high and the contamination from protein or RNA was very low. In fact, the ratio of absorbance values (A1/A2) is very close to 1.8 as a result of the isolation III study shows that there is no contamination.
Conclusions: The isolation and spectrophotometric study results of the peGFP-N1 plasmid showed that this plasmid is suitable and usable in terms of impurity and contamination risk. By making use of the results of this study, it will support the studies in which this plasmid can be used in an appropriate gene carrier system.
References
- 1. Chiang CL, Sung CS, Wu TF. et al. Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells. J. Chromatogr B Analyt Technol Biomed Life Sci. 2005; 822(1-2): 54-60. doi: 10.1016/j.jchromb.2005.05.017
- 2. De Toro M, Garcillán-Barcia MP, De La Cruz F. Plasmid diversity and adaptation analyzed by massive sequencing of Escherichia coli plasmids. Microb. Spectr. 2014; 2: 219–235. doi: 10.1128/microbiolspec. PLAS-0031-2014
- 3. Higuchi Y, Kawakami S, Hashida M. Strategies for in vivo delivery of siRNAs: recent progress. BioDrugs 2010; 24(3):195-205. doi: 10.2165/11534450-000000000-00000.
- 4. Kav AB, Benhar I, Mizrahi I. A method for purifying high quality and high yield plasmid DNA for metagenomics and deep sequencing approaches. J. Microbiol. Methods 2013; 95: 272–279. doi: 10.1016/j.mimet.2013.09.008
- 5. Kim EJ, Park TG, Oh YK. et al. Assessment of siRNA pharmacokinetics using ELISA-based quantification. J Control Release 2010; 143:80-87. doi:10.1016/j.jconrel.2009.12.004
- 6. Klindworth A, Pruesse E, Schweer T. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next- generation sequencing-based diversity studies. Nucleic Acids Res. 2013; 41: e1. doi: 10.1093/nar/gks808.
- 7. Sentchilo V, Mayer AP, Guy L. et al. Community-wide plasmid gene mobilization and selection. ISME J. 2013; 7: 1173–1186. doi: 10.1038/ismej.2013.13
- 8. Shan Z, Wu Q, Wang X. et al. Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles. Anal Biochem. 2010; 398(1): 120-2. doi: 10.1016/j.ab.2009.11.006.
- 9. Stadler J, Lemmens R, Nyhammar T. Plasmid DNA purification. J Gene Med. 2004; 6 (1): S54-66. doi: 10.1002/jgm.512.
- 10. Stewart EJ. Growing unculturable bacteria. J. Bacteriol. 2012; 194: 4151–4160. doi: 10.1128/JB.00345-12.
- 11. Tan SC, Yiap BC. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology 2009; 1-10. http://dx.doi.org/10.1155/2009/574398
- 12. Tseng YC, Mozumdar S, Huang L. Lipid-based systemic delivery of siRNA. Adv Drug Deliv Rev. 2009; 61:721-731. doi: 10.1016/j.addr.2009.03.003
- 13. Warburton PJ, Allan E, Hunter S. et al. Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease, using transposon-aided capture (TRACA). FEMS Microbiol. Ecol. 2011; 78: 349–354. doi: 10.1111/j.1574- 6941.2011.01166. x
- 14. Yeung BZ, Lu Z, Wientjes GM. et al. High Sensitivity RT-qPCR Assay of Nonlabeled siRNA in Small Blood Volume for Pharmacokinetic Studies: Application to Survivin siRNA. AAPS J. 2015; 17(6): 1475-82. doi: 10.1208/s12248-015-9812
There are 14 citations in total.
Details
| Primary Language | English |
|---|---|
| Subjects | Health Care Administration |
| Journal Section | Medical Science Research Articles |
| Authors | |
| Publication Date | September 30, 2021 |
| Acceptance Date | September 25, 2021 |
| Published in Issue | Year 2021Volume: 43 Issue: 3 |