ABSTRACT
A high-throughput robotic workstation system was used for double-stranded plasmid DNA template preparation and sequencing reaction setup to streamline the sequencing process in genome projects. All 96-well miniprep kits that were tested provided high quality plasmid DNA suitable for fluorescent DNA sequencing. After quantitation in a 96-well UV spectrophotometer, the plasmid DNA was used as template to automatically set up sequencing reactions. The setup was controlled by spread sheets that were imported into the robotic system. We utilized this integrated system to prepare all necessary shotgun templates for our contributions to a number of large-scale genome projects as well as a full-length cDNA sequencing project.
Subject(s)
Genome , Robotics , Sequence Analysis, DNA/methods , Arabidopsis/genetics , Bacteriophage M13/genetics , Genome, Bacterial , Genome, Fungal , Genome, Viral , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/geneticsABSTRACT
A new procedure for the PCR amplification of unknown DNA sequences adjacent to a known sequence is described. The required but not readily available second primer sequence in the unknown DNA sequence is obtained by creating an overhanging restriction site in the unknown sequence to which a double-stranded oligonucleotide adaptor of known sequence is ligated.
Subject(s)
Base Sequence , DNA/chemistry , Polymerase Chain Reaction/methods , Blotting, Southern , DNA Restriction Enzymes/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides/geneticsABSTRACT
A new procedure for the photochemical functionalization and the subsequent nonradioactive labeling of synthetic oligonucleotides with psoralen derivatives was developed where a double-stranded poly(A-T) tail is attached to the 5'- or 3'-end of the oligonucleotide to be labeled. The double-stranded poly(A-T) tail is covalently crosslinked by psoralen molecules which carry reactive thiol or amino groups for the attachment of labels. A NH2-specific terbium chelate exhibiting long-lived fluorescence was attached to the functional groups of the intercalated psoralen molecules. Oligonucleotides substituted in this way hybridize readily and can be sensitively detected by time-resolved fluorescence measurements.
Subject(s)
Ficusin , Oligonucleotides/analysis , Terbium , Base Sequence , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Ficusin/chemistry , Fluorescence , Molecular Sequence Data , Oligonucleotides/chemistry , Spectrometry, Fluorescence , Temperature , Terbium/chemistry , Time FactorsABSTRACT
The Cyclotherm instrument is a functionally fully equivalent but inexpensive alternative to commercial instruments for automated polymerase chain reaction (PCR). It can be rebuilt under conditions of a biochemical laboratory for less than +1000. A Peltier element is used for heating and cooling of the reaction vials and the temperature and timing of the PCR cycles are controlled by a BASIC program in a SHARP PC 1600 low cost computer.
