It took scientists more than twenty years after the first DNA sequencing technology was discovered to sequence the entire human genome; yet our own cells complete this task every time our bodies produces a daughter cell. So to achieve the goal of real time DNA sequencing, Pacific Biosciences had the idea to spy on Mother Nature as she goes to work copying DNA. Now, the company's commercial device planned to be on the market in 2010, promises to be 20,000 times faster than current second generation technology, with turn around time of about ten minutes rather than ten days. Chief Technology Officer Steve Turner says in four to five years, new technologies promise to rocket this technology forward even further, making it will possible to sequence an entire human genome in fifteen minutes, on a chip that costs less than 100 dollars.
DNA polymerase is a natural enzyme that constantly makes copies of DNA. DNA is a double stranded chain, and when the two strands are separated, it's possible to recreate one based on the other. That's because DNA consists of only four building blocks, or nucleotides. Each nucleotide only pairs 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, the other side reads TTAACCGG. Polymerase unzips a DNA chain and as long as there are nucleotides to be had, it grabs them and makes a copy of the chain.
Second generation DNA sequencing techniques use polymerase
as a reagent, throwing it away after reading only a few nucleotides in the
sequence. Turner says one of his company's main objectives was to achieve what they call
Single Molecule Real Time DNA Sequencing (SMRT
DNA sequencing) in which they keep the polymerase around longer. One the main problems with watching the
polymerase is knowing how to see the nucleotides. Second generation
techniques attach a different color fluorophore molecule to each type of nucleotide,
lighting them up like Christmas lights. But a whole chain of these lights
creates too much background noise to see the individuals and their order on the chain.
Pacific Biosciences overcame this first challenge by attaching the fluorophores to a part of the nucleotide that is naturally cleaved off by the DNA polymerase. It diffuses away with 100% efficiency in a totally natural process, and keeps the background noise to a low minimum.
To illuminate the individual nucleotides as they are attached to the chain, the company has developed a SMRT chip- a strip of metal with thousands of small wells in it (see image). At the bottom of each well is a polymerase, resting on a glass bottom, and latched onto a strand of DNA which it 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 the background noise from those unattached nucleotides. Engineers shine a laser through the glass bottom, but the light does not come up through the well. This uses the same concept found in your 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 cut in it, which are large enough to let visible light escape, but too small for microwaves to pass through. Similarly, the wavelength of the laser light is too large to pass up through into the wells. But there is an evanescent penetration volume 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, unlike second 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 and only takes ten minute to turn around from the time the sequencing finishes. In addition, the Pacific Biosciences it has three times the read time - or the length of a DNA strand that is read sequentially before the process stops. Normally this is shortened by an overwhelming background noise.
Considering that the technique utilizes a lense and a single-photon CCD array to collect the light, Turner says he hopes DNA sequencing will come to be considered a type of medical imaging in the next ten years.
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