Automated DNA Sequencing

Advances in fluorescent labelling chemistry have led to the development of high-throughput automated sequencing techniques. Essentially, most systems involve the use of dideoxynucleotides labelled with different fluorochromes (often referred to as dye terminators). The advantage of this modification is that since a different label is incorporated with each ddNTP, it is unnecessary to perform four separate reactions. Therefore, the four chain-terminated products are run on the same track of a denaturing electrophoresis gel. Each product with its base-specific dye is excited by a laser and the dye then emits light at its characteristic wavelength. A diffraction grating separates the emissions which are detected by a chargecoupled device (CCD) and the sequence is interpreted by a computer. The advantages of the techniques include real-time detection of the sequence.
In addition, the lengths of sequence that may be analysed are in excess of 500 bp. Capillary electrophoresis is increasingly being used for the detection of sequencing products. This is where liquid polymers in thin capillary tubes are used, obviating the need to pour sequencing gels and requiring little manual operation. This substantially reduces the electrophoresis run times and allows high throughput to be achieved. A number of large-scale sequence facilities are now fully automated, allowing the rapid acquisition of sequence data. Automated sequencing for genome projects is usually based on cycle sequencing using instruments such as the ABI PRISM 3700 DNA Analyzer. This can be formatted to produce simultaneous reads from 384-well cycle sequencing reaction plates. The derived nt sequences are downloaded automatically to databases and manipulated using a variety of bioinformatics resources.