The components of the PyroMark Q96 ID System are designed to make your research workflow straightforward and efficient. Each step is supported by software, kits, reagents, and sample preparation instrumentation that are optimized for Pyrosequencing.
The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi), in a quantity equimolar to the amount of incorporated nucleotide.
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.
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.
Detection of tri- and tetra-allelic SNPs can be difficult with commonly used methods. This series of Pyrograms illustrates the ease of Pyrosequencing based detection of a tri-allelic SNP (red outline). C, T and G are serially dispensed in the Pyrosequencing reaction and only the incorporated nucleotides will elicit a signal peak. The result is a different peak pattern for homozygous samples of each allele (upper three Pyrograms) or compound peak patterns for heterozygous samples (lower three Pyrograms).
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.
Linearity of methylation quantification by Pyrosequencing. PCR products from varying mixtures of unmethylated genomic DNA and methylated DNA (EpiTect Control DNAs) were analyzed by Pyrosequencing. A tight correlation between the known percentage of methylated DNA in the mixtures (squares) and the methylation percentage reported by Pyrosequencing (triangles) was observed (r2 = 0.9962). The graph represents the quantification of methylation at a single CpG site in the p16 gene.
The PyroMark Q96 ID manages all steps necessary to rapidly sequence and analyze up to 96 samples. Simply design the necessary assay and run files, load your samples and reagents, and walk away. The PyroMark Q96 ID dispenses reagents and nucleotides to each well with precision and detects emitted light signals with sensitive CCD sensors.
By processing 96 samples in a single run, the PyroMark Q96 ID combines analysis versatility with high throughput. The PyroMark Q96 MD explands this throughput with a robotic module that enables hands-free processing of ten 96-well plates. New assay design can be done with the PyroMark Q24, which offers the same analysis versatility on a smaller throughput scale.
PyroMark Q96 Software and PyroMark supplementary software are user-friendly interfaces granting access to assay design, run setup, and powerful data analyses of the obtained results. The software are driven by dropdown menus that ensure the correct selection of parameters and analysis modes for any assay, enabling new users to perform Pyrosequencing runs almost immediately.
The PyroMark Q96 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 can be carried out for up to 96 samples in parallel and takes only a few minutes.
Analysis of multiple contiguous CpG sites. Methylation at nine independent CpG sites (highlighted in gray) is quantified in a single Pyrosequencing run. Position-specific information in the context of an analyzed sequence presents broad sequence methylation patterns. Note the built-in quality control sites (highlighted in yellow) consisting of cytosines converted to thymines, demonstrating full bisulfite conversion of the treated DNA.
Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori.