The PyroMark Q24 MDx Vacuum Workstation enables conversion of PCR products into the single-stranded DNA needed as template for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process is carried out for 24 samples in parallel and takes only a few minutes.
Though small in size, PyroMark Q24 Advanced manages all steps necessary to rapidly analyze up to 24 samples. Simply load your samples and reagents, upload your run file, and walk away. The instrument dispenses reagents and nucleotides to each well with precision. Light signals emitted are detected by 24 CCD sensors — one sensor per well — thereby eliminating signal crossover.
Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-thio triphosphate (dATPαS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP), since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues, the complementary DNA strand is elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace.
The components of the PyroMark Q24 Advanced system are designed to make the Pyrosequencing workflow straightforward and efficient. Each step, from assay design to PCR amplification and preparation of sequencing templates, is supported by software, kits, reagents, and sample prep instrumentation optimized for Pyrosequencing.
ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by CCD sensors and seen as a peak in the raw data output (Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.
The PyroMark Q24 MDx is designed as a stand-alone instrument, which makes it easy to place anywhere in the lab. Data are stored on the instrument hard drive and can be viewed on the instrument screen during a run. Additionally, all files are stored on the supplied USB stick, giving the user the flexibility to analyze data on any computer with PyroMark Q24 MDx Software installed.
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.