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Color-blind method opens new doors in DNA sequencing

A "color-blind" method of fluorescence detection developed by researchers at Baylor College of Medicine (BCM) and Rice University could open new doors that would take DNA sequencing to the patient's bedside, the doctor's office and even the scene of a crime or a battlefield.

"We could eventually do direct detection of a DNA sequence from native DNA" without manipulation now performed in the laboratory, said Dr. Michael L. Metzker, assistant professor in the BCM Human Genome Sequencing Center and adjunct assistant professor of chemistry at Rice. "We could make sequencing portable and do it faster."

The research appears this week in the journal Proceedings of the National Academy of Sciences. In the paper, Metzker, Rice University Professor Robert Curl and colleagues from BCM and Rice describe a new way of doing DNA sequencing that could be more accurate than current methods.

DNA in the nucleus of every human cell is made of long chains of building blocks called nucleotides. DNA is made up of just four types of nucleotides ?referred to as A, C, G and T ?and is organized in such a way that A binds T and G binds C, forming a double helical structure. Each person's genome consists of a unique ordering of some 3 billion base pairs, and 'DNA sequencing' refers to the process scientists use to read out the order of those nucleotides.

In sequencing, scientists first extract DNA from the nuclei of cells and through a painstaking series of bacterial cloning and/or polymerase chain reaction (PCR) steps, reduce its length to a manageable size of thousands of nucleotides. Using natural replicating enzymes, the DNA is tagged with four fluorescent dyes, each corresponding to a particular nucleotide. This tagging process, called Sanger sequencing, results in smaller DNA fragments, which are then separated base-by-base. Because the DNA fragments are tagged with dyes, they glow when they are struck by laser light to determine the order of one's DNA seque nce.

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 or commercial development.

Others who participated in the research include 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, all of Rice, and Ernest K. Lewis, Wade C. Haaland, and Graham B. I. Scott, all of BCM.


Source:Baylor College of Medicine

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