Analysis of plasmid DNA damage induced by melanin with capillary electrophoresis.
Dilute linear poly(N-isopropylacrylamide) (PNIPAM) in Tris-Mes-EDTA (TME) buffer was used as sieving matrix for capillary electrophoresis (CE) of plasmid DNA and plasmid topological isomers induced by melanin in uncoated capillary. At the optimized condition of 0.1% (w/v) PNIPAM in TME buffer, base line separation of the plasmid DNA ladder (2-12 kbp) was achieved within 15 min. Three positive clones with inserts of 468, 1147 and 1566 bp can be distinguished from the plasmid pUC 18 vector within 13 min. The migration order of the plasmid topological isomers in the dynamic coating matrix was confirmed by the enzymatically prepared and UV-induced plasmids. The covalently closed circular form appeared firstly, followed by the linear plasmid form and then the open circular form. The effect of bacterial melanin obtained from Pseudomonas maltophilia AT18 on plasmid pUC 18 was investigated by CE in uncoated capillary in vitro. Plasmid pUC 18 incubated with either melanin or copper ions alone sustained little DNA damage. The combination of melanin with Cu(II) can cause the plasmid pUC 18 conformational changes from covalently closed circular form to open form. Understanding the damage effect of melanin with copper ions on DNA would be important for the melanin-related application, such as photoprotective antioxidant in protecting the skin from cancer, pathophysiology research in clinic. The investigation of melanin induced plasmid conformational changes by CE in uncoated capillary also revealed that the application of the dynamic coating matrix could be extended to the study of plasmid conformational changes in other plasmid-based biological technologies.
Yu
Sheng-Bing
SB
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China.
Geng
Jing
J
Zhou
Ping
P
Feng
Ai-Rong
AR
Chen
Xiang-Dong
XD
Hu
Ji-Ming
JM
D000180:Acrylic Resins / Q000737:chemistry; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D004249:DNA Damage; D004269:DNA, Bacterial / Q000737:chemistry; D015245:Deoxyribonuclease BamHI / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D008543:Melanins / Q000633:toxicity*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry* / Q000187:drug effects*; D011549:Pseudomonas / Q000737:chemistry
D000180:Acrylic Resins; D004269:DNA, Bacterial; D007202:Indicators and Reagents; D008543:Melanins; C052970:poly-N-isopropylacrylamide; D003300:Copper; D004492:Edetic Acid; D015245:Deoxyribonuclease BamHI