Method of the Month — Pyrosequencing
February 2022
Pyrosequencing is a cost-effective and time-efficient method for sequencing single-stranded DNA. The method uses a cocktail of enzymes and nucleic acids to determine the sequence of the DNA as it is being replicated in real time. Light is released upon addition of each nucleotide to the growing DNA strand, and the detection of this light is what yields the sequence of the DNA.
To understand pyrosequencing, one must first understand the details of DNA replication. DNA must first be “unzipped” into a single strand to expose the adenines, thymines, cytosines, and guanines to which a complementary base will pair (A to T, G to C). To permanently incorporate the base into the growing DNA strand, a new bond is formed along the phosphate backbone, and a molecule called pyrophosphate is released (for a more detailed explanation, click here). The key is in the ratio; for one base being added, one pyrophosphate is released. The release of pyrophosphate is then detected through a chain reaction of enzymes which ends with the release of light. Now this base pair addition can be quantified because each reaction is 1:1.
The first step in a pyrosequencing assay is to isolate the desired DNA strand and unwind it to single-stranded DNA. This single-stranded DNA is then anchored to a plate with the base pairs facing upward into a solution. For pyrosequencing to work, this solution must contain three enzymes: DNA polymerase, ATP sulfurylase, and luciferase. A fourth enzyme, apyrase, is often added to “clean up” the solution in between each round of base pairs (more on this later).
Once the DNA strand is anchored, a single type of dNTP is added (either A, T, G, or C). DNA polymerase adds this dNTP to the growing complement strand, and pyrophosphate (PPi) is released. ATP sulfurylase turns the pyrophosphate into ATP. In this solution, the only thing for ATP to bind to is the luciferase enzyme. This enzyme releases light upon binding to ATP, and this light can be detected by a sensor. The detection can be calibrated so that the intensity of the light indicates how many times a base pair is added.
The connection between light signals and sequencing a genome may be a bit confusing, so let’s consider a scaled-down example.
If you have a DNA target strand with a sequence AACG, and you add ten thymine (T) molecules, two would bind (one at each A). Two pyrophosphates would be released, and two luciferases would light up and be detected. Apyrase (the cleanup enzyme) would then get rid of the other eight thymines, and you would add the next base (let’s pick guanine). Ten guanines are added, one binds to the C, and one luciferase shines. When two luciferases light up versus only one, the difference in signal strength shows how many base pairs were added. This process is repeated hundreds of times across the target strand until a profile of light signals versus base pair identity is created. This is what shows the sequence of the DNA.
Overall, the method of pyrosequencing is a very quick, effective method for sequencing DNA. It does not require gel electrophoresis, nor does it require large-scale replication of DNA in chain-termination PCR (both of these are required for Sanger Sequencing). Pyrosequencing has become a staple technique for clinicians and researchers alike, and it certainly deserves its distinction.
For more about pyrosequencing, check out the following links:
