PyroMark Q96 Tests

用于配合PyroMark Q96 ID和PyroMark Q96 MD进行基因特异的序列变异和甲基化


  • 检测并定量分析癌症相关基因的突变
  • 定量分析不同基因的单个或多个CpG位点
  • 确定特定区域的CpG位点甲基化状况
  • 序列背景提供检测的内置参照
PyroMark HFE (96)

Cat. No. / ID: 972442

For detection of variants in the HFE gene (2 x 96 reactions on PyroMark Q96 MD, 1 x 96 reactions on PyroMark Q96 ID, 8 x 24 reactions on PyroMark Q24)
CpG Prader-Willi
CpG p16
PyroMark APOE、PyroMark HFE、PyroMark MTHFR、PyroMark Q96 CpG LINE-1、PyroMark Q96 CpG MGMT、PyroMark Q96 CpG MLH1、PyroMark Q96 CpG p16和PyroMark Q96 CpG Prader-Willi检测适用于分子生物学应用,不适用于疾病的诊断、预防或治疗。

Product Details

PyroMark Q96 Tests适用于高度可靠的突变及SNP定量检测和CpG位点甲基化分析,经优化配合PyroMark Q96 MD和/或PyroMark Q96 ID使用。产品名带有“Q96”的产品应与PyroMark Q96 MD和/或PyroMark Q96 ID仪器配套使用,PyroMark HFE和PyroMark MTHFR等其他产品也可配合PyroMark Q24使用(PyroMark APOE除外)。


PyroMark Q96 Tests适合用于准确检测CpG的甲基化状态,例如PyroMark Q96 CpG p16 Test(参见“ PyroMark p16 analysis”)。
See figures


PyroMark Q96 Tests配合PyroMark Q96 MD和/或PyroMark Q96 ID Systems采用焦磷酸测序技术准确定量突变(或SNP)和进行甲基化分析。这些产品包括PCR引物和一个测序引物。


在焦磷酸测序之前,形成了一个生物素标记的PCR产物,该产物结合到覆盖链酶亲和素的琼脂糖凝胶珠上。这些胶珠会被真空仪器捕捉并彻底清洗,随后变性,得到适合测序的单链DNA。PyroMark Q96 Tests提供预制的甲基化分析试剂,用于焦磷酸测序。每个分析含有用于扩增生物素标记的PCR产物所需的引物,也含有焦磷酸测序反应所需的引物。


PyroMark Q96 Tests能够高度准确的定量检测甲基化和进行突变(或SNP)分析,用于多个研究领域,包括癌症和表观遗传学研究。

Supporting data and figures


Y chromosomal STR analysis using Pyrosequencing technology.
Edlund H; Allen M;
Forensic Sci Int Genet; 2009; 3 (2):119-24 2009 Jan 6 PMID:19215881
DNA methylation profiles in monozygotic and dizygotic twins.
Kaminsky ZA; Tang T; Wang SC; Ptak C; Oh GH; Wong AH; Feldcamp LA; Virtanen C; Halfvarson J; Tysk C; McRae AF; Visscher PM; Montgomery GW; Gottesman II; Martin NG; Petronis A;
Nat Genet; 2009; 41 (2):240-5 2009 Jan 18 PMID:19151718
Amelogenin sex determination by pyrosequencing of short PCR products.
Tschentscher F; Frey UH; Bajanowski T;
Int J Legal Med; 2008; 122 (4):333-5 2008 Mar 20 PMID:18351373
Identification of mammal species using species-specific DNA pyrosequencing.
Karlsson AO; Holmlund G;
Forensic Sci Int; 2007; 173 (1):16-20 2007 Feb 28 PMID:17331687
More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA.
Malmström H; Svensson EM; Gilbert MT; Willerslev E; Götherström A; Holmlund G;
Mol Biol Evol; 2007; 24 (4):998-1004 2007 Jan 25 PMID:17255122


Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
Does the PyroMark CpG LINE assay target mouse transposons as well?

The PyroMark CpG LINE-1 assay is specific for human DNA and was not tested on mouse DNA.  Mouse LINE-1 elements differ slightly in sequence and may not be detected.



Can the PyroMark Q96 CpG LINE assay be used with an ID system?
The PyroMark Q96 CpG LINE-1 assay can only be used with a PyroMark Q96 MD system because the PyroMark Q96 ID instrument does not have a sensitive enough camera. For the Pyromark Q24 there is a dedicated PyroMark Q24 LINE-1 assay.
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
What is the amplicon length of the PyroMark CpG LINE assay?
The amplicon length of the PyroMark CpG LINE-1 PCR product is about 146 bp. for PyroMark Q24 and PyroMark Q96 MD.
Does the PyroMark LINE assay target all LINE sequences?
The PyroMark assay is designed to cover LINE-1 retrotransposable elements in the genome. Those differ slightly in sequence and it might be that not all are picked up by our LINE-1 assay. The intention of the assay is to cover as many as possible of the LINE-1 sequences.
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
What region of LINE gets targeted by the assay for PyroMark Q96 MD or PyroMark Q24?

The PyroMark Q96 CpG LINE-1 for PyroMark Q96 MD is designed to target four CpG sites in positions 331 to 305 (GenBank accession no X58075). The PyroMark Q24 CpG LINE-1 assay for PyroMark Q24 targets three CpG sites in positions 331 to 318 (GenBank accession no X58075). The target region is human LINE-1 transposon DNA consensus sequence.

Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
How many CpG sites are analyzed by the PyroMark CpG LINE assay?
The PyroMark Q24 LINE-1 assay covers 3 CpG sites and the LINE-1 assay for PyroMark Q96 MD covers 4 sites.
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878