We use the latest technology, capillary electrophoresis, to generate the best possible data for our customers. Capillary separation is used for analysis of many types of biological molecules and is capable of providing high resolution of large molecules. Compared with the use of polyacrylamide gels as a separation medium, capillaries allow faster separation, thus shorter run times, and the ability to automate processes since the capillaries can be refilled on the instrument rather than a new polyacrylamide gel needing to be poured. Samples are "loaded" onto the capillaries via a process known as electrokinetic injection, which relies on the charge of the molecule.
For semi-automated Sanger DNA sequencing, fluorescently labeled fragments are produced using fluorophores attached to the chain terminating di-deoxynucleotides. Modern systems use four differently coloured fluorophores so that all four nucleotides can be identified in a single capillary. The capillary is filled with a denaturing polymer material and the capillaries are heated in an oven to encourage full denaturation of the DNA strands and migration of fragments according to their length in a high voltage electrophoresis system. As the fragments near the anode electrode, they pass through the detector cell where a powerful laser beam causes the fluorophores attached to the fragments to fluoresce. This fluorescence is detected by a CCD camera and sent to a computer for analysis.
When sequencing bacterial plasmids, isolation of DNA from the bacterial cells is a critical first step. Whilst the amount of material required for a single reaction is not much, the purity of the material is very important. Plasmid DNA mini-preps usually yield enough material, but mini-prep methods can vary enormously in the quality of plasmid DNA obtained. Quick “boiling” methods or home-made kits do not produce DNA of a quality that is sufficient for obtaining long, accurate reads. In order to generate DNA that is of an appropriate purity, we utilise commercial DNA purification kits (Promega, Wizard SV 96(TM)) combined with robotic extraction (Qiagen, Bio-Robot 9600(TM)). This approach consistently produces high purity plasmid DNA that is suitable for use in the DNA sequencing reaction. For isolation of larger amounts of plasmid DNA, we perform plasmid maxi-preps using Invitrogen Pure-link HiPure(TM) kits and plasmid midi-preps using Promega PureYield(TM) kits. This DNA is routinely used by customers for various research purposes, including mammalian cell transfection (the DNA isolated via the midi-prep kits is endotoxin free, making it especially suitable for this), but can, of course, also be sequenced.
Analysis of DNA Fragments
Related to DNA sequencing, analysis of fluorescent DNA fragments also utilises the genetic analysis instruments we have. However, run conditions are different and a fluorescent size marker is included with each sample to provide an internal sizing standard. This results in very accurate relative size information that allows fragments differing in size by only a single nucleotide to be distinguished. Though we do not perform the PCR reactions that produce the fluorescent fragments, we do undertake all subsequent steps in the preparation and analysis of such fragments using our in-house technology.