Most sequencing today is done with one laser and optics to separate the dyes into the four colors, blue, green, yellow, and red. A common problem with the technique is that the color of light emitted by the dyes is similar. Even with complex computer programs to assist in deciphering the signals, this "cross-talk" between the dyes results in subtle variations that can cause nucleotides to be miscalled.
The new method developed at BCM and Rice, called pulsed multiline excitation, uses four lasers, each matched to a particular dye. PME enables the researchers to take advantage of the entire visible spectrum, eliminating the problem of cross-talk between dyes, said Metzker.
Because there are four lasers, scientists can manipulate the system so that each dye gives the same intensity of fluorescent signal, eliminating the need for further software processing to yield readable sequence information.
"Genome sequencing, by its very nature, is a process that begs for precision, and the number of mistakes that can be tolerated is extremely low," said Curl, University Professor, the Kenneth S. Pitzer-Schlumberger Professor of Natural Sciences and professor of chemistry. "Our new method does away with identification problems altogether, because the imaging is very clean."
Metzker said, "We have built a highly sensitive instrument for the measuring of fluorescence, because PME gives brighter signals and collects more of that signal by eliminating the need for a prism to separate the light into colors."
Metzker is also seeking to develop a chip-based imager than could be used in his overall project on sequencing-by-synthesis (SBS), which is funded by the National Human Genome Research Institute. SBS could lead to the ability to sequence an individual's own genome rapidly and inexpensively.
Metzker and the major developers of this technology filed a patent on PME in 2001, which has been exclusively licensed to LaserGen f
Source:Baylor College of Medicine