Development of an automated procedure for fluorescent DNA sequencing.
We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.
Wilson
R K
RK
Division of Biology, California Institute of Technology, Pasadena 91125.
Chen
C
C
Avdalovic
N
N
Burns
J
J
Hood
L
L
D001331:Automation; D004274:DNA, Recombinant / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation; D009710:Nucleotide Mapping / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D012371:Robotics
D004274:DNA, Recombinant; D005456:Fluorescent Dyes