PyroMark Q48 Autoprep
For automated Pyrosequencing with integrated template preparation for advanced methylation, mutation and SNP quantification
PyroMark Q48 Autoprep integrates fully automated template preparation into the Pyrosequencing protocol. The new design and software double the throughput of PyroMark Q24 Advanced, while keeping the improved performance, making it ideally suited for functional studies, as well as for verification and validation of large numbers of samples from NGS and array experiments.
The PyroMark Q48 Autoprep is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
Advanced technology, software and chemistry for long and reliable sequence runsPyroMark Q48 Autoprep features improved chemistry and instrument operation algorithms that significantly increase assay read length and accuracy in base calling, mutation analysis and methylation quantification compared to PyroMark Q96 and PyroMark Q24 systems. Assay read length in previous systems was limited by background peaks and reduced light signals in the sequencing reaction. The updated PyroMark “Advanced” chemistry and algorithms reduce this background, thereby increasing read length and reliability. Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single reaction.
Increased throughput for more SNP and mutation assays per runMultiple Primer Dispensation (MPD) is a strategy to increase the capacity of automated sequencing primer dispensation in case more assays are needed than cartridge reservoirs are available. The PyroMark Q48 Autoprep Primer Cartridge has 3 reservoirs, but if more assays are needed per disc, the primers can be mixed and filled into the same reservoir of the primer cartridge. After template preparation, primer mixes are dispensed into the wells automatically. Since only one PCR template is present in each well, only the corresponding sequencing primer will bind to the template. All other primers will stay in solution but will not bind (see The principle of multiple primer dispensation). Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.
Compatibility of assays among different PyroMark platformsPreviously designed Pyrosequencing assays are readily compatible with the new PyroMark Q48 Autoprep instrument and the “Advanced” chemistry. Data indicate that the same mutation frequencies and methylation quantification results are obtained when an assay is run on the various PyroMark platforms (see Compatibility among PyroMark platforms for methylation analysis and Compatibility among PyroMark platforms for mutation analysis). The cross-platform compatibility is also independent of the distance of the analyzed site away from the sequencing primer. This is particularly important when analyzing multiple sequence variations in a single run, which is typical for complex mutation assays or methylation analysis of consecutive CpG sites.
New disc design enables magnetic template preparation without cross contaminationPyroMark Q48 Discs are specially designed to automate template preparation and Pyrosequencing in the same instrument without manual user interaction. All buffers used for template preparation are efficiently removed from the sample and the disc during the run without the risk of any cross-contamination from well-to-well of one run or from disc-to-disc between subsequent runs (see Excluding well-to-well and disc-to-disc cross-contamination).
The PyroMark Q48 Autoprep uses proven real-time sequence-based Pyrosequencing technology for detection and quantification in genetic analyses and epigenetic methylation studies. The system can analyze up to 48 samples simultaneously. An easy-to-use, automated protocol prepares single-stranded DNA samples without manual interaction from the user. This protocol uses magnetic streptavidin-coated Sepharose beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand. Annealing of sequencing primers can be automated for up to four different sequencing primers. If more sequencing primers are used, the primers can be manually added to the single-stranded DNA samples.
Steps of the Pyrosequencing reaction:Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer. The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure "Principle of Pyrosequencing – step 1").
Step 2: 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 (see figure "Principle of Pyrosequencing – step 2").
Step 3: 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 (see figure "Principle of Pyrosequencing – step 3").
Step 4: Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure "Principle of Pyrosequencing – step 4").
Step 5: 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 (see figure "Principle of Pyrosequencing – step 5").
A run file is created using the PyroMark Q48 Advanced software, and is transferred to the instrument via a USB drive or network connection. Reagents, nucleotides and buffers are loaded into the PyroMark Q48 Autoprep cartridges, according to the volumes indicated on the touchscreen. The biotinylated PCR product and magnetic streptavidin-coated Sepharose beads are loaded into the wells of the Q48 Disc, and the disc and an absorber strip are placed into the instrument. Preparation of the single-stranded template, along with sequencing primer annealing are now completely automated, and require no additional user interaction. Up to 3 separate sequencing primers or Multiple Primer Dispensation (MPD) mixes can be dispensed automatically. Optional, manual primer addition further increases the number of different assays per run.
Pyrosequencing is increasingly important for research applications in a variety of disciplines. Whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock or polymorphisms in forensic samples of mitochondrial DNA, PyroMark platforms enable powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.
PyroMark Q48 Autoprep Software, installed on a PC, enables comprehensive analysis of your results. The software contains 4 analysis modes – AQ, SNP, CpG and SEQ. The AQ mode can be used for a variety of quantification studies of mutations such as SNPs and InDels. It is suitable for analyzing single and multivariable positions, as well as di-, tri- and tetra- allelic mutations. The SNP mode provides genotype analysis of SNPs and InDels. The CpG mode enables methylation analysis of single or multiple CpG or CpN sites and provides a built-in control for the bisulfite treatment. The SEQ mode is used for base calling of unknown sequences.
The PyroMark Q48 Autoprep Software is user-friendly and intuitive and provides convenient and improved tools for run analysis. If a problem occurs during the run, or if the system detects an inconsistency with an assay, the software provides specific warning information for each individual well. A "Warning Info" button gives access to additional information about the warning along with recommendations for troubleshooting and preventing its occurrence in subsequent assays.
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