Novel and More Expedient Method of Determining the Nucleotide Sequence of DNA Molecules Using Fluorescent Detection

A human cell contains in its chromosomes approximately 2 meters of DNA. The human genome is the base sequence of that DNA that encodes for the organism in a linear triplet code of 3-letter words composed from the four DNA bases, adenine, thymine, guanine and cytosine (A, T, G, C). Human DNA contains a string of approximately 3 x 109 bases, and the objective of the Human Genome Project is to read out this entire sequence. DNA-based medical and diagnostic applications will require rapid, cheap readout of small portions of the genome, a few hundred bases in length.Conventional DNA sequencing technology uses the Sanger process in which DNA polymerase enzymes are used in four separate reactions to make multiple copies of a template DNA strand in which the growth process has been arrested at each occurrence of an A, in one set of reactions, and a G, a C and a T respectively in the others. This produces a series of copies of different lengths, and the lengths signal the positions along the template strand at which each of the four bases occurs. The copies are ordered according to length using gel electrophoresis and the order of the four bases is read directly from the length ordering.While the Sanger technique is very powerful particularly in an automated form, it is nevertheless much too slow and too expensive to be used to sequence the entire human genome, and also too slow and expensive to be used subsequently in diagnostic applications. Therefore there is a continuing search for novel new technologies which will be significantly faster and cheaper.The ideal approach to DNA sequencing would be to somehow read the sequence directly, base-by-base along the strand. The only such approach yet suggested is to chew back along a strand, one base at a time, using an exonuclease enzyme, and identify each base as it is produced. However, if multiple DNA strands are digested simultaneously, to generate sufficient signal for easy detection, the multiple polymerase reactions rapidly get out of phase and information is garbled. The only potentially feasible approach is to digest a single DNA strand - but then the challenge is to detect and identify single nucleotide molecules. This is extremely difficult and years of research have yet to produce a workable technology.Researchers at Arizona State University have developed a concept under which enzymes can be made to "march in step", allowing them to essentially build a synthetic replica of an unknown DNA molecule in exact sequence. Coupling this technique with suitable flow control and detection systems (also developed and patented at ASU) allows an extraordinarily rapid, accurate and efficient determination of the nucleotide sequence of a DNA molecule.With this latest iteration of this invention, the researchers have added a fluorescent-based detection method that adds a further element of automation and ease of analysis.This invention has obvious benefits not only in the area of genomic sequencing, but also in the area of gene related diagnostic and therapeutic applications as well.