Next generation sequencing is all the rave in genetics laboratories nowadays, it seems like everybody and their mums’ able to get the sequence of the DNA they’re interested in. DNA sequencing wasn’t always as easy to do as it is now. Sanger sequencing was invented in 1977, a slow, laborious and expensive process that was used for over 25 years. It was only in the mid 90s that next generation sequencing was being developed, and it was only brought into use around 2004.
SMRT (Single Molecule Real Time) sequencing is a highly advanced next gen process, which was developed by Pacific Biosystems. It was first used in a paper published in Science in 2009, by Eid et al.
SMRT sequencing uses DNA polymerase, an enzyme that synthesises a new DNA strand out of individual nucleotides, using an existing single stranded DNA molecule as a template. If you can watch which nucleotides the DNA polymerase incorporates, you will be able to find out the sequence of nucleotides as the DNA polymerase processes along the template DNA.
To visualise which nucleotide is being incorporated, the nucleotides are labelled with a fluorophore. Fluorophores are small molecules that absorb light and re-emit it at a new wavelength. The four different nucleotide bases (G, A, T and C) are each labelled with a fluorophore of a different wavelength, so they can be told apart.
The process itself is an elegant exploitation of the natural workings of DNA polymerase, shown below.
As the DNA polymerase lines up the correct nucleotide base with the complementary base of the template DNA strand, the fluorophore shines in what’s called a fluorescent pulse. When the pyrophosphate group bound to the nucleotide is cleaved off by the DNA polymerase, the pyrophosphate and the attached fluorophore diffuse away, ending the fluorescent pulse. The energy from the breaking of that bond is used to form the phosphodiester bond between the current nucleotide and the previous nucleotide.
The DNA polymerase then translocates the template DNA along to the next base, and waits for the complementary nucleotide to diffuse into its active site, which will then provide the next fluorescent pulse in the sequence.
It’s not quite as simple as that though. DNA polymerases don’t synthesise DNA at a constant rate, they start and stop. This means to get the sequence of nucleotides, only one DNA polymerase must be observed in one spot. This is achieved with structures called zero mode waveguides (ZMWs).
The ZMW is a tiny little well, only 100 nm across. The reason they are this small is that only one DNA polymerase will occupy each ZMW. The DNA polymerase is fixed the base of the ZMW with a special layer of biotinylated polyethylene glycol, which stops the DNA polymerase floating away.
Tens of thousands of individual ZMWs are arrayed on a chip. The ZMWs are so small that only very small wavelengths of light are able to enter them, so low wavelength lasers are used to illuminate the array. The resulting fluorescent pulse sequences from the DNA polymerases are recorded by a sophisticated “high-multiplex confocal fluorescence detection system”.
The technology is able to keep track of 3000 ZMWs at once and the DNA polymerases are able to read 5,000 to 8,000 bases in just over an hour. At that rate, you could sequence the entire human genome in a day with just 14,000 ZMWs, though they’d need to upgrade the detection system. Mind you, at $700,000 for Pacific Biosystem’s state of the art RS II SMRT sequencing machine, it’d burn a bit of a hole in your pocket. However, the relentless advance of technology and science will hopefully cut that cost down in the next decade or two.
All in all, SMRT sequencing is an amazing piece of technology, and the whole process is summarised quite nicely in this little video by Pacific Biosystems.
This article was based on Eid et al. (2009) Science. 323:133-138. It is available on the NCBI, here.