USE OF NON-CROSS-LINKED POLYACRYLAMIDE FOR 4-COLOR DNA-SEQUENCING BY CAPILLARY ELECTROPHORESIS SEPARATION OF FRAGMENTS UP TO 640 BASES IN LENGTH IN 2 HOURS

Four-color DNA cycle sequencing was performed on an M13mp18 template u sing dye-labeled primers, Sequencing fragments were separated by capil lary 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 formati on of secondary structures in the sequencing fragments, generating a s equence that is free of compressions without the use of strongly denat uring gels. The use of high temperatures also increases the separation rate compared with room-temperature operation, Fragments up to 640 ba ses are separated in less than 2 h.