Researchers at Baylor College of Medicine (BCM) and Rice University have developed a"colour-blind" method of fluorescence detection which 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.
Dr. Michael L. Metzker, assistant professor in the BCM Human Genome Sequencing Centre and adjunct assistant professor of chemistry at Rice, says the technique could eventually allow direct detection of a DNA sequence from native DNA without the complex manipulation presently performed in the laboratory, it would make sequencing portable and faster.
In the research 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. The paper appears this week in the journal Proceedings of the National Academy of Sciences.
DNA in the nucleus of every human cell is made of long chains of building blocks called nucleotides, DNA sequencing' refers to the process scientists use to read out the order of those nucleotides. It is made up of four types of nucleotides – A, C, G and T – and is organized so 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.
In sequencing, scientists 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.
Then by 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 which helps to determine the order of the DNA sequence.
Sequencing today is usually done with one laser and optics to separate the dyes into the four colours, blue, green, yellow, and red, and the problem with the technique is that the colour of light emitted by the dyes is too similar. Complex computer programs help to decipher the signals, but cross-talk, subtle variations between the dyes, can cause nucleotides to be miscalled.
This new method developed at BCM and Rice, called pulsed multiline excitation (PME), eliminates this problem by using four lasers, each matched to a particular dye and enables researchers to take advantage of the entire visible spectrum.
The four lasers allow scientists to 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.
They 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 colours.
Curl, University Professor, the Kenneth S. Pitzer-Schlumberger Professor of Natural Sciences and professor of chemistry says that the very nature of genome sequencing is a process that demands precision, and mistakes cannot be tolerated, the new method does away with identification problems altogether, because the imaging is very clean.
Dr. Metzker is also hoping 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.
He and the major developers of this technology have filed a patent on PME in 2001, LaserGen has an exclusive license for the commercial development of this technology.
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.