Use of non-cross-linked polyacrylamide for four-color DNA sequencing by capillary electrophoresis separation of fragments up to 640 bases in length in two hours

Abstract

Four-color DNA cycle sequencing was performed on an M13mp18 template using dye-labeled primers. Sequencing fragments were separated by capillary electrophoresis at 60 degrees C and at an electric field of 150 V/cm. The sieving medium was 5%T, non-cross-linked polyacrylamide in 7 M urea. The use of high temperature for the separation reduces formation of secondary structures in the sequencing fragments, generating a sequence that is free of compressions without the use of strongly denaturing gels. The use of high temperatures also increases the separation rate compared with room-temperature operation. Fragments up to 640 bases are separated in less than 2 h.