Automated magnetic preparation of DNA templates for solid phase sequencing.
An integrated protocol for solid-phase DNA sequencing using a robotic work station is described involving magnetic separation of DNA and analysis of the sequencing product by electrophoresis with automated detection of the fluorescently labeled fragments. The method, which is based on magnetic beads in combination with streptavidin-biotin technology, can be used for sequencing both genomic and plasmid DNA. The DNA template is obtained by the polymerase chain reaction (PCR). Protocols to prepare five and ten immobilized samples is described, giving 10 and 20 single-stranded templates, respectively. The magnetic purification steps are performed in a microtiter plate and this allows for an integrated scheme involving a subsequent procedure for automated primer annealing and sequencing reactions. Here, the procedure is examplified by direct genomic sequencing of DNA in blood sample from a human immunodeficiency virus (HIV)-infected patient and a cloned human antibody DNA fragment using fluorescently labeled sequencing primers.
Wahlberg
J
J
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Holmberg
A
A
Bergh
S
S
Hultman
T
T
Uhlén
M
M
D001331:Automation; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012371:Robotics; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004247:DNA