Pacific Biosciences, a four year old company out of Menlo Park, California that managed to raise $120 million in venture capital last year (a large sum of money, had we not been conditioned to counting in trillions in the last few weeks), has just released details about its new DNA sequencing technology. Called single-molecule real-time sequencing (SMRT®), the technology promises genetic testing at under $200 per person, according to Bloomberg.
The company explains how SMRT® allows “for the first time, the observation of natural DNA synthesis by a DNA polymerase as it occurs,” and how the sequencing is done:
Our approach is based on eavesdropping on a single DNA polymerase molecule working in a continuous, processive manner. Distinguished by its long reads, short run times, and high quality sequence data with less effort and cost, SMRT DNA sequencing promises to be a transformative technology that will enable a new paradigm in genomic analysis.
PacBio’s SMRT technology is built upon two key innovations that overcome major challenges facing the field of DNA sequencing:
The SMRT chip, which enables observation of individual fluorophores against a dense background of labeled nucleotides by maintaining a high signal-to-noise ratio, and Phospholinked nucleotides, which produce a completely natural DNA strand through fast, accurate, and processive DNA synthesis
DNA sequencing is performed on SMRT chips, each containing thousands of zero-mode waveguides (ZMWs). Utilizing the latest geometries available in semiconductor manufacturing, a ZMW is a hole, tens of nanometers in diameter, fabricated in a 100 nm metal film deposited on a silicon dioxide substrate. Each ZMW becomes a nanophotonic visualization chamber providing a detection volume of just 20 zeptoliters (10-21 liters). At this volume, the technology detects the activity of a single molecule among a background of thousands of labeled nucleotides.
The ZMW provides a window for watching DNA polymerase as it performs sequencing by synthesis. Within each chamber, a single DNA polymerase molecule is attached to the bottom surface such that it permanently resides within the detection volume. Phospholinked nucleotides, each type labeled with a different colored fluorophore, are then introduced into the reaction solution at high concentrations which promote enzyme speed, accuracy, and processivity. Due to the small size of the ZMW, even at these high biologically relevent concentrations, nucleotides occupy the detection volume only a small fraction of the time. In addition, visits to the detection volume are fast, lasting only a few microseconds due to the very small distance that diffusion has to carry the nucleotides. The result is a very low background.
When DNA polymerase incorporates complementary nucleotides, the enzyme holds each nucleotide within the detection volume for tens of milliseconds—orders of magnitude longer than the amount of time it takes a nucleotide to diffuse in and out of the detection volume. During this time, the engaged fluorophore emits fluorescent light whose color corresponds to the base identity. Then, as part of the natural incorporation cycle, the polymerase cleaves the bond that previously held the fluorophore in place and the dye diffuses out of the detection volume. Following incorporation, the signal immediately returns to baseline and the process repeats.
Unhampered and uninterrupted, the DNA polymerase continues incorporating bases at a speed of tens per second. In this way, the SMRT approach produces a completely natural long chain of DNA in minutes. Simultaneous and continuous excitation and detection occurs across all of the thousands of ZMWs in the SMRT chip in real time. Researchers at Pacific Biosciences have demonstrated this approach has the capability to produce reads thousands of nucleotides in length.