Individual Base Pairs

Individual base pairs, being added to a DNA undergoing replication, have been monitored in real time using DNA polymerase and fluorescently tagged nucleotides for the first time.

Polymerase is the enzyme which replicates DNA in living organisms. In vitro it does this at a rate of about 700 base-pairs per second. This is far too fast a process for current equipment to watch at the level of a single base, much less determine which of the four canonical bases (A, C, T, and G) is being incorporated at that moment.

But at Harold Craighead's lab at Cornell the addition of single bases can be detected (but not yet identified) as it happens. First a polymerase is stuck to the bottom of a channel of a microfluidic device. Then single-stranded DNA floats by and the polymerase begins a replication session by sequentially adding complementary base units to the DNA strand. A burst of fluorescence is emitted each time the polymerase incorporates a base, so like a series of flash bulbs going off the synthesis can be watched in real time (contact Mathieu Foquet, 607-255-6286, mf37@cornell.edu).

Ideally the fluorescence would employ four different colors, one for each type of base. The Cornell group so far can muster two colors. With four-color fluorescence this microfluidic process might help to speed up greatly the task of genome sequencing. The new results were presented at the meeting of the AVS Science and Technology Society in Denver, November 3-8; paper NS/BI/MoA5)