Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes.
Standard visualization of nucleic acids by electron microscopy requires the use of special spreading techniques. The most common method takes advantage of the formation of a complex between negatively charged nucleic acid molecules and a positively charged monolayer film of proteins or cationic agents. Here, we describe an alternative protocol for the rapid visualization of DNA by electron microscopy based on the complexes formed when nucleic acids are exposed to buffers containing polyamines in the presence of sodium chloride. This procedure has been devised for the detection and analysis of large DNA molecules, such as yeast artificial chromosomes, but can be applied to DNA molecules of small size as well. The formation of DNA-polyamine complexes stabilizes large DNA molecules in solution and prevents shearing. This property allows large DNA molecules to remain intact after passage through microcapillaries used in the generation of transgenic mice by microinjection of fertilized eggs.
Montoliu
L
L
Division of Molecular Biology of the Cell I, German Cancer Research Centre, Heidelberg.
Bock
C T
CT
Schütz
G
G
Zentgraf
H
H
D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D015825:Chromosomes, Fungal / Q000235:genetics*; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure*; D004271:DNA, Fungal / Q000737:chemistry / Q000648:ultrastructure; D016521:Electrophoresis, Gel, Pulsed-Field; D008854:Microscopy, Electron; D011073:Polyamines / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012965:Sodium Chloride / Q000737:chemistry
D004271:DNA, Fungal; D011073:Polyamines; D012965:Sodium Chloride; D004247:DNA