DNA polymerase is a natural enzyme that constantly makescopies of DNA. DNA is a double stranded chain, and when the two strands areseparated, it’s possible to recreate one based on the other. That’s because DNAconsists of only four building blocks, or nucleotides. Each nucleotide onlypairs up with one other: adenine (A) pairs with thymine (T), and guanine (G)pairs with cytosine (C). Thus, if one side of the chain reads AATTGGCC, theother side reads TTAACCGG. Polymerase unzips a DNA chain and as long as thereare nucleotides to be had, it grabs them and makes a copy of the chain.
Second generation DNA sequencing techniques use polymeraseas a reagent, throwing it away after reading only a few nucleotides in thesequence. Turner says one of his company’s main objectives was to achieve what they callSingle Molecule Real Time DNA Sequencing (SMRTDNA sequencing) in which they keep the polymerase around longer. One the main problems with watching thepolymerase is knowing how to see the nucleotides. Second generationtechniques attach a different color fluorophore molecule to each type of nucleotide,lighting them up like Christmas lights. But a whole chain of these lightscreates too much background noise to see the individuals and their order on the chain.
Pacific Biosciences overcame this first challenge byattaching the fluorophores to a part of the nucleotide that is naturallycleaved off by the DNA polymerase. It diffuses away with 100% efficiency in atotally natural process, and keeps the background noise to a low minimum.
To illuminate the individual nucleotides as they areattached to the chain, the company has developed a SMRT chip- a strip of metalwith thousands of small wells in it (see image). At the bottom of each well is apolymerase, resting on a glass bottom, and latched onto a strand of DNA whichit will unzip and begin to copy. Base pairs fly around the surface of the chip,but only a few at a time dip down into the well. This immediately minimizes thebackground noise from those unattached nucleotides. Engineers shine a laser through the glass bottom, but thelight does not come up through the well. This uses the same concept found inyour microwave oven. You don’t want those microwaves flying around the kitchen,but you want to see what’s cooking. So the door of the oven has small holes cutin it, which are large enough to let visible light escape, but too small formicrowaves to pass through. Similarly, the wavelength of the laser light is toolarge to pass up through into the wells. But there is an evanescent penetrationvolume of only a few zeptoliters (10-21). This illuminates the polymerase, but not the space around it where the nucleotides are floating. And when a nucleotide is bound to the DNA cahin, it releases a burst of light.
The Pacific Biosciences technique uses no reagent, unlikesecond generation sequencing methods which use a tremendous amount of reagent(it would take two semitrailers full to sequence an entire human genome). It’s 20,000 times faster andonly takes ten minute to turn around from the time the sequencing finishes. Inaddition, the Pacific Biosciences it has three times the read time -or the length of a DNA strand that is read sequentially before the processstops. Normally this is shortened by an overwhelming background noise.
Considering that the technique utilizes a lense and asingle-photon CCD array to collect the light, Turner says he hopes DNAsequencing will come to be considered a type of medical imaging in the next tenyears.