2004-01-22 17:49:12WuYung123
利用原子力顯微鏡掃瞄DNA 2
Visualisation of DNA by AFM 2 (利用原子力顯微鏡掃瞄DNA 2)
(based on Hansma and Laney, 1996)
Mica is a common surface for AFM imaging of DNA, Even moving DNA molecules as short as 300bp (79 to 1057 bp(~25 to350nm)) can sometimes be imaged by AFM in aqueous conditions. There are two ways of preparing DNA samples for AFM imaging on mica:
1. drying DNA onto mica: deposit DNA solution onto freshly split mica, rinsed with water, and dried thoroughly. The DNA dried onto mica can then be imaged either in air or in fluid.
2. imaging NDA solutions on mica: DNA in aqueous solutions can be imaged on mica in the AFM even without drying, if the solution contains Ni(II)
Salts of divalent or multivalent inorganic cation can increase the amount of DNA bound to the mica, e.g. Mg, Ca, Ba, Co, Ni, Zn, Cr, La and Zr. In contrast, monovalent cations such as K decrease the binding of DNA to mica. Mg or Ca do not bind DNA in solution tightly enough to mica for DNA imagining without drying. Ni is the only cation previously known to bind DNA to mica tightly enough for AFM imaging without drying. Clear images of DNA on mica were obtained with Ni, Co and Zn ions in solution. With Mn, DNA only bind weakly to mica and it move during imaging. For solution with Cd, Hg, Mg or Ca does not bind to mica tightly enough for imaging in liquid. For DNA solution with Cu, AFM images showed lumps instead of DNA strands. The amount of DNA bound is also dependent on the DNA concentration.
DNA binding to mica correlates well with the ionic radius of the transition metal cation present in the buffer. When the ionic radius is 0.74A or less, DNA binds tightly to mica even allowing imaging in fluid. Both mica and DNA have negatively charged surface. Thus, divalent cations probably bind DNA to mica by bridging the charges. The correlation of DNA binding with cationic radius could be due to the steric requirement of either the DNA or the mica. Research on mica suggests that the mica structure, not the DNA structure, is the reason. The observed differences between Mg and Ni, Co or Zn do not seem to come from difference in their binding to DNA. Cations ban bind to either the phosphate backbone or the bases of DNA. Cations that stabilize the DNA double helix by bridging phosphate groups rise the DNA’s melting temp. Perhaps Mg can bind DNA to mica electrostatically by ross-bridging the negatively charged DNA and mica. A steric view of the sample suggests that the spacing of charges on DNA may affect binding. Phosphate groups on DNA have a spacing of 3A, whereas the mica lattice spacing is 5A. Thus the optimal binding of DNA to mica might occur with1ion/nm DNA, where every third DNA base could bind to every second mica site. With more than 1 ion/nm, DNA binding to mica might decrease. DNA binding is also pH dependence, where there is ~25% less DNA on the mica at 7 than at pH8.
When the DNA solution does not contain Ni but the mica has been rinsed with Ni, some of the DNA molecules move on the mica surface in such a way that they can be imaged in changing position in the AFM. Co can also be used instead of Ni for movies.
In conclusion, DNA in solutions containing divalent transition metal cations binds to mica tightly enough for AFM imaging if the ionic radius of the transition metal cation is .69 to .74A. One possible explanation is that small transition metal cations can fit into the cavities above the recessed hydroxyl groups on the mica surface, although it is also possible due to they hydrated cations.
(based on Hansma and Laney, 1996)
Mica is a common surface for AFM imaging of DNA, Even moving DNA molecules as short as 300bp (79 to 1057 bp(~25 to350nm)) can sometimes be imaged by AFM in aqueous conditions. There are two ways of preparing DNA samples for AFM imaging on mica:
1. drying DNA onto mica: deposit DNA solution onto freshly split mica, rinsed with water, and dried thoroughly. The DNA dried onto mica can then be imaged either in air or in fluid.
2. imaging NDA solutions on mica: DNA in aqueous solutions can be imaged on mica in the AFM even without drying, if the solution contains Ni(II)
Salts of divalent or multivalent inorganic cation can increase the amount of DNA bound to the mica, e.g. Mg, Ca, Ba, Co, Ni, Zn, Cr, La and Zr. In contrast, monovalent cations such as K decrease the binding of DNA to mica. Mg or Ca do not bind DNA in solution tightly enough to mica for DNA imagining without drying. Ni is the only cation previously known to bind DNA to mica tightly enough for AFM imaging without drying. Clear images of DNA on mica were obtained with Ni, Co and Zn ions in solution. With Mn, DNA only bind weakly to mica and it move during imaging. For solution with Cd, Hg, Mg or Ca does not bind to mica tightly enough for imaging in liquid. For DNA solution with Cu, AFM images showed lumps instead of DNA strands. The amount of DNA bound is also dependent on the DNA concentration.
DNA binding to mica correlates well with the ionic radius of the transition metal cation present in the buffer. When the ionic radius is 0.74A or less, DNA binds tightly to mica even allowing imaging in fluid. Both mica and DNA have negatively charged surface. Thus, divalent cations probably bind DNA to mica by bridging the charges. The correlation of DNA binding with cationic radius could be due to the steric requirement of either the DNA or the mica. Research on mica suggests that the mica structure, not the DNA structure, is the reason. The observed differences between Mg and Ni, Co or Zn do not seem to come from difference in their binding to DNA. Cations ban bind to either the phosphate backbone or the bases of DNA. Cations that stabilize the DNA double helix by bridging phosphate groups rise the DNA’s melting temp. Perhaps Mg can bind DNA to mica electrostatically by ross-bridging the negatively charged DNA and mica. A steric view of the sample suggests that the spacing of charges on DNA may affect binding. Phosphate groups on DNA have a spacing of 3A, whereas the mica lattice spacing is 5A. Thus the optimal binding of DNA to mica might occur with1ion/nm DNA, where every third DNA base could bind to every second mica site. With more than 1 ion/nm, DNA binding to mica might decrease. DNA binding is also pH dependence, where there is ~25% less DNA on the mica at 7 than at pH8.
When the DNA solution does not contain Ni but the mica has been rinsed with Ni, some of the DNA molecules move on the mica surface in such a way that they can be imaged in changing position in the AFM. Co can also be used instead of Ni for movies.
In conclusion, DNA in solutions containing divalent transition metal cations binds to mica tightly enough for AFM imaging if the ionic radius of the transition metal cation is .69 to .74A. One possible explanation is that small transition metal cations can fit into the cavities above the recessed hydroxyl groups on the mica surface, although it is also possible due to they hydrated cations.