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PyroMark Q24 Advanced Reagents

For advanced methylation and mutation quantification in long sequence runs using Pyrosequencing
  • Advanced technology, software, and chemistry for long sequence runs
  • Quantitative methylation analysis at consecutive CpG or CpN sites
  • Improved quantification of sequence variations at any sequence position

PyroMark Q24 Advanced Reagents feature advanced Pyrosequencing chemistry to provide even better real-time sequence-based detection and quantification than before. PyroMark Q24 Advanced Reagents are optimized for use with PyroMark Q24 Advanced Systems and are highly suited for analyzing any kind of sequence variation, particularly DNA methylation at CpG or CpN sites, complex mutations, or for de novo sequencing applications such as microbial typing. For long-read Pyrosequencing runs which may require larger volumes of nucleotides, such as de novo sequencing or methylation analyses we recommend using the PyroMark Q24 Advanced CpG Reagents.
Cat No./ID: 970902
PyroMark Q24 Advanced Reagents (4 x 24)
$386.00
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Enzyme mix, substrate mix, nucleotides, and buffers for Pyrosequencing assays; intended for use with PyroMark Q24 Advanced
Cat No./ID: 970922
PyroMark Q24 Advanced CpG Reagents (4 x 24)
$424.00
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Enzyme mix, substrate mix, nucleotides, and buffers for CpG and long read Pyrosequencing assays; intended for use with PyroMark Q24 Advanced

PyroMark Q24 Advanced Reagents are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


Performance
Longer Pyrosequencing runs and improved accuracy
PyroMark Q24 Advanced features improved chemistry and instrument operation algorithms that significantly increase the assay read length and accuracy of the base calling functionality, enabling easy analysis of long de novo sequencing runs. Assay read length was previously limited by background peaks and reduced light signals in the sequencing reaction. The updated chemistry and algorithms of PyroMark Q24 Advanced reduce these background peaks, 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 PyroMark Q24 Advanced reaction (see figure Long de novo sequencing runs).

Improved methylation analysis at any position
PyroMark Q24 Advanced enables improved methylation quantification in long sequence runs at any sequence position. Previously, analysis of methylation sites further away from the sequencing primer could be uncertain, but now with longer read lengths and higher accuracy, methylation quantification is highly reliable throughout the entire sequencing run (see figure Analysis of 16 CpG sites in a long sequence run).

Improved sequencing accuracy in homopolymers
Bisulfite conversion in DNA methylation analysis leads to frequent poly T stretches in the nucleotide sequence, and analysis of CpG sites directly after such T homopolymers has previously been challenging due to uncertain quantification of the light signal at these sites. The increased accuracy of PyroMark Q24 Advanced enables reliable quantification of CpG methylation behind and even within a stretch of 8 T nucleotides (see figure Improved methylation quantification in homopolymers).

Analysis of mutations over long sequences
PyroMark Q24 Advanced also provides reliable quantification of multiple polymorphisms in a single assay. Since single nucleotide polymorphisms (SNPs) and other mutations are often not located close to one another, traditional Pyrosequencing chemistry usually requires separate assays for each mutation site to be analyzed. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of more than one mutation or SNP in the same run (see figure Quantitative mutation analysis in long sequence runs).
Principle
Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents feature updated Pyrosequencing chemistry to enable analysis of complex mutations, methylation analysis in epigenetic research, resistance typing, and microbial identification. These reagents are optimized for use with PyroMark Q24 Advanced Systems.
Procedure
Fast and easy sample preparation 
Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.
Applications
Pyrosequencing is becoming 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, the PyroMark Q24 Advanced enables 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.

Product comparison
  PyroMark Q24 Advanced PyroMark Q24
Throughput 1–24 samples 1–24 samples
Running volume 25 µl 25 µl 
PCR requirements 5–10 µl
(~0.5–3 pmol of product)
5–10 µl
(~0.5–3 pmol of product)            
Read length (assay- and
sequence-dependent)
10–140 bp or more 10–80 bp
Application software PyroMark Q24 Advanced SW
(requires firmware 1.5.6903 or higher)
PyroMark Q24 SW 2.0
Software functionality SEQ (de novo sequencing)
CpG/CpN methylation
SNP
AQ
SQA (de novo sequencing)
CpG methylation
AQ/SNP 
 
Main applications Complex mutation analysis
Epigenetics (CpG and CpN analysis)
Resistance typing and microbial ID  
Mutation analysis
Resistance typing 
 
Compatible reagents PyroMark Q24 Advanced Reagents
PyroMark Q24 Advanced CpG Reagents  
PyroMark Q24 Gold Reagents 
Sensitivity 2% mutation
98% wt  
2% mutation
98% wt  
Important: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents cannot be used with PyroMark Q24 Software. PyroMark Gold Q24 Reagents cannot be used with PyroMark Q24 Advanced Software. Using the incorrect reagent and software combination will result in incorrect reagent dispensations and lead to inaccurate results.

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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0003
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0004
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0005
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0006
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0007
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0008
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0009
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0010
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0011
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0012
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0013
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Application Notes (1)
A Pyrosequencing method for the forensic identification of tissue source using QIAGEN’s PyroMark Q24 Advanced system.
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