Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides

As the human genome project proceeds, various types of DNA analysis tools a re required for life sciences and medical sciences including DNA diagnostic s. For example, a small DNA sequencer for sequencing a short DNA is require d for bed-side DNA testing as well as DNA analysis in a small laboratory. H ere, a new handy DNA sequencing system (pyrosequencer) based on the detecti on of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The ke y point for the miniaturized: DNA sequencer is to make a deoxynucleotide tr iphosphate (dNTP) delivery system small and inexpensive. it has been realiz ed by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most ca ses, more than 40 bases can be successfully sequenced. A homopolymeric regi on, which can not be easily sequenced by a conventional gel-based DNA seque ncer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was e asily achieved with this small instrument.