New procedure of high-voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids

✍ Scribed by T.V. Kutateladze; V.D. Axelrod; V.G. Gorbulev; S.N. Belzhelarskaya; R.M. Vartikyan

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 586 KB
• Volume: 100
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90121-0

✦ Synopsis

Fractionation

of nucleic acids and their fragments with polyacrylamide gel has been widely applied in sequencing of nucleic acids. Although the conditions of electrophoresis for this purpose have previously been suggested, we have found that polyacrylamide gel electrophoresis at 5000 V (100 V/cm) is possible and effective. An apparatus consisting of a horizontal thermostated plate is used to remove the heat which was formed during the electrophoretic process. The techniques for loading samples on the horizontal thin gel and the procedure for high-voltage gel electrophoresis are described and illustrated by the fractionation of the spleen phosphodiesterase partial digest of tRNA$' as well as by the RNA synthesis by RNA polymerase from E. coli with poly[d(A-T)]

as template in the presence of "terminator," 3'-O-methyluridine 5'-triphosphate. This same technique was used for electrophoresis of oligonucleotides on acetylcellulose and was incorporated into a two-dimensional system which was demonstrated by fingerprinting of the guanylo-RNase digest of tRNATr* from baker's yeast. In the third part of the article a simple technique for the electric trapping of nucleic acids or their fragments from a slab gel on a DEAE-paper sheet is presented.