Method of the Month: Single Molecule Real-Time Sequencing
October 2022
The next method in this sequencing series is known as single-molecule real-time sequencing. This technique is a third-generation technology.
Nearly ten years ago Pacific Biosciences, a biotechnology company that manufactures gene sequencing products, produced a new method of DNA sequencing that would change the future of the company and the scientific community. In 2011, they invented Single Molecule Real Time Sequencing (SMRT Sequencing). This is a third-generation, parallelized single-molecule DNA sequencing method that allows scientists to observe DNA synthesis while it occurs. This technology uses a zero-mode waveguide (ZMW) as a tool for synthesis. Pacific Biosciences (PacBio) commercialized SMRT sequencing in 2011. In 2012 this process could read about 2500 to 2900 bases. In 2014, with newer technology and better chemistry, the process could read 500 million to 1 billion bases per SMRT Cell. Many advantages come from using SMRT sequencing including amplification without the use of a polymerase chain reaction (PCR), which can occasionally become faulty, and visualization of the process of DNA synthesis while it is happening.
The critical factor in SMRT sequencing is the build of the machine that is used to hold the reaction. SMRT cells hold several ZWM chambers. These chambers are 70 nanometers wide and 100 nanometers deep with an open top and a glass bottom. A light will shine through the bottom of the glass with a camera. However, since the nanostructure is so small, the wavelength of light is too big to pass all the way through the chamber, so it stops about 20 or 30 nm into the nanostructure, creating a space called the 20 zeptoliter detection volume- the only volume in this chamber where light is able to pass.
Now to begin with the actual DNA sequencing details. In these ZMW chambers, a DNA molecule shaped like a hairpin will be attached at the glass bottom. On the ends of the hairpin are ligated adapters, which are chemically synthesized fragments of DNA. These attach to the actual template fragments of DNA, the “sides” of the hairpin which are the relevant replication fragments. After the adapters are attached to the bottom of the nanostructure, primers and DNA polymerase are added to the mix. However, to begin this replication, we need nucleotides so the polymerase can start pairing bases. So, scientists dye each base pair (adenosine, guanine, thymine, and cytosine) with a fluorescent dye and linker, each with their own color- red, green, blue, or yellow- and add it to the ZMW chamber. Then, DNA polymerase can begin replication. Polymerase attaches to the primer and starts laps around the hairpin-like DNA structure creating extended newly formed molecules of DNA. It is because of the structure of the template DNA molecule that the polymerase can move faster and with little to no error in replication, consequently giving larger base pair reads.
As replication occurs, and base pairs are added, the dye assigned to the nucleotide reflects the color it is assigned because of the light passing through the glass at the bottom of the ZMW. The color assigned to the base completely lights up the chamber, creating what's called a color pulse. That flash is what the camera under the glass captures. So, scientists are able to see which base pair gets connected based on the color and repetition of pulses after replication begins.
Results at the end of this process are new copied segments of DNA, which are called sub-reads, and a visualization of the entire process. In contrast to its second-generation version, Illumina sequencing, SMRT sequencing allows DNA polymerization, the synthesis of monomer nucleotides to polymer DNA, to occur much quicker and with much longer base pair reads. As science and technology are constantly advancing and evolving, we are able to see the progress being made every day in the field of genetics.
For more information on Single Molecule Real-time Sequencing, check out the following links!
