PyroMark Q24 Tests

For analyzing sequence variation or methylation of specific genes using the PyroMark Q24 Instrument

Features

Detection and quantification of different mutations in a single run
Flexible software for post-run analysis of adjacent mutations
Quantification of individual or multiple CpG sites in different genes
Estimation of global CpG methylation in defined regions of interest
Sequence context provides built-in control of the assay

Useful Resources

(EN) - PyroMark HFE Handbook (EN) - PyroMark MTHFR Handbook (EN) - PyroMark Q24 CpG LINE-1 Handbook

PyroMark Q24 CpG MLH1 (4 x 24)

Cat. No. / ID: 970022

Methylation detection assay: PCR primers and sequencing primer for quantification of methylation level in region –209 to –181 from the transcription start site in the MLH1 gene

Assay

PyroMark Q24 Test

PyroMark Q96 Test

Type

CpG MLH1

CpG p16

CpG LINE-1

CpG MGMT

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

Product Details

PyroMark Q24 Tests are a range of assays suitable for highly reliable, quantitative mutation and CpG methylation analysis and are optimized for use with the PyroMark Q24. Tests designated with 'Q24' are optimized for use with the PyroMark Q24, while others such as PyroMark HFE and PyroMark MTHFR can be used with the PyroMark Q96 ID and PyroMark Q96 MD as well. For more tests for these instruments, see PyroMark Q96 Tests.

Principle

PyroMark Q24 CpG LINE-1, PyroMark Q24 CpG MGMT, PyroMark Q24 CpG MLH1, and PyroMark Q24 CpG p16 enable highly reliable methylation results. In addition, a built-in control for bisulfite treatment improves accuracy and reproducibility of the results.

Procedure

Prior to Pyrosequencing, a biotinylated PCR product is generated. Biotinylated PCR products are bound to streptavidin-coated Sepharose beads. The beads are then captured with the vacuum tool on the PyroMark Vacuum Workstation, and throroughly washed, generating single-stranded DNA suitable for sequencing. PyroMark Q24 Tests are predesigned assays for methylation analysis by Pyrosequencing. Each test includes the primers necessary for generating the biotinylated PCR product, as well as the primer for the Pyrosequencing reaction.

Applications

PyroMark Q24 Tests enable highly accurate, quantitative methylation and mutation (or SNP) analysis applicable in several fields of research, including cancer and epigenetics.

Resources

Publications

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

FAQ

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

The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.

FAQ ID -2827

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

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

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.

-1

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.

-1

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

The amplicon length of the PyroMark CpG LINE-1 PCR product is about 146 bp. for PyroMark Q24 and PyroMark Q96 MD.

-1

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.

-1

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

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.

-1

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

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

The PyroMark Q24 LINE-1 assay covers 3 CpG sites and the LINE-1 assay for PyroMark Q96 MD covers 4 sites.

-1

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

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

PyroMark Q24 uses Pyrosequencing technology for mutation analysis and provides a built-in quality control in each run. By sequencing nucleotides flanking the mutation of interest, the researcher gets confirmation that the assay was made at the correct position. In addition, blank dispensations are included in the assay providing a negative control of the run.

FAQ ID -2094

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