30. Separation of rice chromosome-sized DNAs by orthogonal-field-alternation gel electrophoresis (OFAGE)


Laboratory of Radiation Genetics and Chemical Mutagenesis, Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113 Japan

Because the size of the eukaryote's genomic DNA is very large, a method for separation of full size chromosome DNA molecules needed to be developed. Orthogonal-field-alternation gel electrophoresis has been reported to allow separation of DNA fragments up to at least 2,000 kilobase pairs (kb) long (Carle and Olson 1984). Using this technique, we fractioned six ethidium bromide-stained bands of rice genomic DNA in a running gel, which were apparently chromosome-sized DNAs.

To avoid physical shearing during DNA preparation, we developed a very mild process called gel supported sampling. Nuclei were excised from the embryos of dry rice seeds (Oryza sativa L.) cv. 'Akihikari', and quickly mixed with low- melting temperature agarose at 37°C. To release chromatin from the nuclei and digest the nuclear protein, sample gel slices were incubated in a buffer containing sodium N-lauroylsarcosinate and proteinase K for 48 hr at 50°C.

After incubation, gel slice was cut to a suitable size and subjected to electrophoresis. Six bands were observed from the running gel, as shown in Fig. 1. Comparing them to Yeast (SHY 3 strain) chromosome-sized DNA run simultaneously as a marker, we found that the band which had the lowest molecular weight migrated at almost the same rate as yeast chromosome V or VIII, indicating a molecular weight of approxiamtely 700 kb. Three bands near the loading site migrated much more slowly than that of the yeast chromosome IV. Therefore, it is believed that they have molecular weights of more than 2,000 kb.

If DNAs were physically sheared during preparation, we would fail to obtain discrete bands using electrophoresis. Therefore, it is reasonable to suppose that the six separated bands obtained in this study are chromosome-sized molecules of rice DNA.


Fig. 1. A. Ethidium bromide-stained ORAGE agarose gel after 18 hr's running with a pulse frequency of 60 sec and both directions supplied by 300V.The sample applied to the center part of the gel was rice DNA prepared in this study and those on the two sides were yeast (SHY 3strain) chromosome-sized DNA.

B. Densitometric scan of A's negative film. Arrows show the separate bands in the gel.



Carle, G. F. and M. V. Olson 1984. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acid Res. 12: 5647-5664.