Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori.
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.
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).
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.
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.
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.