Four Lasers Used for Color-Blind DNA Sequencing

Increasing the speed and accuracy of DNA sequencing and decreasing its cost are necessary for it to become a routine part of individualized medicine. Traditionally, identifying the order of bases in a sequence has required the labeling of pieces of DNA with four colors of fluorescent dyes that usually are excited by one laser. This method is limited in speed and accuracy because it requires significant software steps to account for inefficient excitation of dyes from a single laser, for crosstalk between the dyes' emission and for excitation maxima and losses in signal.

Researchers at Baylor College of Medicine and at Rice University, both in Houston, have developed a technique that overcomes the limitations of traditional sequencing by detecting fluorescence in a color-blind manner. It excites fluorescent dyes using four lasers -- with wavelengths of 399, 488, 594 and 685 nm -- pulsed sequentially in a way that correlates to the response of a photomultiplier tube. The investigators also identified a set of dyes with well-separated emission and excitation maxima that match well with the lasers' wavelengths.

In the April 12 issue of PNAS, they show that the sequencing method decreases software steps, excites all the dyes efficiently and eliminates crosstalk. It also reduces signal loss because optics are not needed to separate the fluorescence into four colors.

The researchers are working on building microchip devices based on the technique. LaserGen Inc., also in Houston, has licensed the detection method and plans to have a commercial product by 2006.