What is Pyrosequencing?
Pyrosequencing is a method of determining nucleotide sequence for a short piece of DNA and is ideally suited to the detection of mutations in an animal's genetic code. Samples can be blood, tissue or cells collected on a swab.
What are the steps involved in a pyrosequencing assay?
The PyroMark Q24 pyrosequencer in the laboratory
- Samples are collected and sent to the laboratory. For ease of collection we recommend taking mouth swab samples as described here.
- In the laboratory we extract the genomic DNA from the cheek cells present on the swab to both purify and concentrate it.
- We then perform a PCR to amplify the region of DNA surrounding the genetic mutation of interest.
- The PCR product is then purified and a sequencing primer is bound next to the mutation.
- The samples are then placed in the pyrosequencing instrument, which determines the genetic sequence of the mutation.
Typical pyrosequencing result showing heterozygous sequence (deletion/T) at position three
Typical pyrosequencing result showing homozygous wild type sequence (T/T) at position three
Typical pyrosequencing result showing homozygous mutant sequence (deletion/deletion) at position three
What are the advantages of pyrosequencing?
- Pyrosequencing allows us to quickly and accurately determine the sequence of a genetic mutation
- The mutation can be a simple substitution (e.g. feline PKD) or more complicated (e.g. feline blood type genetics). The mutation can even be a large insertion, deletion or inversion.