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.
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.
Zhang
J
J
Department of Chemistry, University of Alberta, Edmonton, Canada.
Fang
Y
Y
Hou
J Y
JY
Ren
H J
HJ
Jiang
R
R
Roos
P
P
Dovichi
N J
NJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D004247:DNA