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New DNA sequencing method is 'color-blind'

by Ruth SoRelle, MPH

Michael Metzker, PhD

Until now, color ruled in the sequencing of DNA.

A technique using a single laser and fluorescent dyes enabled the international consortium of genome researchers to sequence the entire human genome two years early.

However, technology does not stand still, and faster and cheaper is the common goal in the genome sequencing effort. In fact, Francis Collins, MD, PhD, director of the National Genome Research Institute, recently told a Baylor College of Medicine audience that he anticipates it will soon be possible to sequence an individual person's genome quickly and for less than $1,000. While "soon" is an imprecise term, work at Baylor College of Medicine and Rice University in Houston is already advancing the cause of faster and cheaper sequencing with a new technique called pulsed multiline excitation. A report on their work appeared in a recent issue of the journal Proceedings of the National Academy of Sciences.

To understand the new technique developed by Michael Metzker, PhD, assistant professor in the BCM Human Genome Sequencing Center along with Robert Curl, PhD, professor at Rice University (and a Nobel laureate), one must first understand what DNA is and why it is sequenced.

DNA is the genetic material from which our genes and chromosomes are made. It is made up of chemicals called bases - adenine (A), thymine (T), cytosine (C) and guanine (G). The order or sequence in which these bases appear in DNA governs which proteins a particular gene causes the cell to make and, ultimately, how the cell carries out processes necessary to continued existence.

When researchers sequence the DNA, each base is tagged with a particular dye that is different from the dyes tagging the other bases. A single laser excites the fluorescent dyes and they are separated into four colors. From this, the computer "reads" the order in which the bases appear in the DNA strand.

However, the system is not foolproof.

"When you have a single laser and four dyes, the dyes have to be close enough (on the color spectrum) that the furthest dye can still be excited by the laser," said Metzker. Because of that, the fluorescent emission of green can interfere with the blue or the yellow. That can result in problems with reading the sequence.

Pulsed multiline excitation, by contrast, uses four lasers, enabling the dyes to spread across the entire visible spectrum and eliminating the problem of crosstalk, said Metzker.

"Each laser is matched to a particular dye," he said. "A violet laser is matched to a violet dye, etc."

The four lasers make it possible for each dye to give off the same intensity of fluorescent signal. Because of this, no prism to separate the colors is needed and the computer does not have to adjust the data to come up with a readable DNA sequence.

"We have built a highly sensitive instrument for the measuring of fluorescence," said Metzker. In essence, the system is "color blind" because it does not rely on the detection of color to identify a particular base.

"This method is good and a lot of effort has been put into refining it," said Curl, professor of natural sciences at Rice. "But 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. Our new method does away with identification problems altogether, because the imaging is very clean."

"We could eventually do direct detection of a DNA sequence from native DNA" without manipulation now performed in the laboratory, said Metzker, "We could make sequencing portable and do it faster."

That might make DNA sequencing portable, enabling it to be used directly at the patient's bedside, the doctor's office and even the scene of a crime or on a battlefield.

Metzker and the major developers of this technology filed a patent on PME in 2001, which has been exclusively licensed to LaserGen for commercial development.

Others who participated in the research include: Ernest K. Lewis, Wade C. Haaland, and Graham B. I. Scott of BCM and Carter Kittrell, Bruce R. Johnson, Freddy Nguyen, Daniel A. Heller, Matthew J. Allen, Robert R. MacGregor, C. Scott Berger, Lori A. Burns, and Britain Willingham of Rice University.

Funding for this project came from the National Institutes of Health and the National Science Foundation.

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