PyroMark™Q24

PyroMark Q24

A Complete solution for Mutation and Methylation Analysis

• CpG methylation• Allele quantification • Mutation analysis

A Small Smart Affordable Pyrosequencing System with a 24 well format

Pyrosequencing PyroMark™Q24

Pyrosequencing technology offers an established genetic analysis method based on the principles of sequencing by synthesis.

PyroMark Q24, a system running up to 24 samples in parallel.– Pyrosequencing assays are simple, fast and robust with

inbuilt controls – The results are shown in real time on the instrument

display

PyroMark Q24 System

InstrumentVacuum Prep. Workstation

Application software

Reagents

The PyroMark Q24 System

• PyroMark Q24 Instrument• PyroMark Q24 Analysis Software• PyroMark Q24 Vacuum Prep Workstation• Reagents, Consumables and Accessories• PyroMark Q24 Validation Oligo• PyroMark Control Oligo• PyroMark Q24 RUO • Assay DB updated with 69 new CpG

methylation assays

PyroMark™Q24

The system PyroMark Q24 and Vacuum prep workstation

Benefits of PyroMark™Q24

• An extremely easy to use system• Up to 24 samples in parallel

– Control oligo delivered with the system ensures proper installation

– Validation oligo is available to check the performance of the system

• Small Footprint– Takes very little bench space – measuring only H420xW390xD525mm

• Robust– Can be run at ambient temperatures between

15°C and 32°C.• Accurate

PyroMark™Q24

Runs can be set up and analyzed on any computer anywhere, running PyroMark™Q24 Software.

Information is transferred between the instrument and computer via a USB memory stick.

Pyrosequencing Technology

• A non destructive technology that enables further template analysis

• Ideal for paraffin embedded samples

• An extensive database of assays is available via the Biotagebio.com site

Pyrosequencing for the clinic

• Pyrosequencing offers a Gold Standard for genetic testing

1. Reproducible sequence data with built-in QC is most appropriate for a clinical decision

2. Pyrosequencing is easier, faster and cheaper than traditional sequencing

3. Pyrosequencing quantifies the proportion of mutant cells among normal cells

4. In contrast, hybridization assays with differential probes:– require subjective interpretation– have no inherent QC

Vacuum Prep Workstation

The PCR products have to be processed to yield single-stranded DNA to which a sequencing primer can be annealed.

Samples to be analyzed using PyroMark™Q24 should be prepared, using PyroMark™Q24 Vacuum Prep Workstation.

Q24 Vacuum Prep Workstation

• 1-24 post-PCR samples prepared in parallel in less than 15 minutes

• Minimal pipetting

• Actual hands-on time, less than 1 minute

• Plastic waste reduced to a minimum

PyroMark Q24 Software

The software has two analysis modes: • AQ A variety of quantification studies and SNP analysis. • CpG Methylation analysis in multiple consecutive CpG

sites.

• AQ assays and CpG assays can be performed on the same PyroMark™Q24 Plate.

• You can toggle between the analysis modes in the analysis view of the software, by selecting AQ or CpG in the toolbar.

Both modes offer detailed report information that can be exported to html, excel, text and pdf formats.

PyroMarkTM Q24

CpG Methylation

Bisulfite conversion of DNA

PCR Sample Prep

~ 2h ~ 10 min~ 15 min

Pyrosequencing

CpG Methylation Analysis

• Quantitative analysis of multiple consecutive sites

• Flexible assay design

- Forward – reverse/ Upper – lower

- Flexible primer positioning

• Built-in Bisulfite treatment control

• Excellent Performance

- Accuracy

- Precision

- Reproducibility over time

• Confidently discern even small changes in methylation

• Fast results

CpG mode

• Analyze methylation in the presence of SNP:s• Suitable for analysis of fresh-frozen, fixed and

paraffin-embedded specimens • Present deviation from predefined methylation

ranges• Quality assessment of individual sites and

sequence context• Mean methylation for the whole assay shown

– for quick overview of results• Many assays have been validated using

Pyrosequencing technology • 24 samples analyzed in parallel in 1 hour after PCR

A Robust, Accurate System

• Accuracy - Linear response in measured methylation

• Methylation levels are consistent even when using different primers

• Confidently measure the individual degree of methylation in adjacent CpG sites, even at long distances from the sequencing primer

• Easily handles varying methylation levels from site to site

• Detects site variation reproducibly

CpG methylation studies with PyroMarkTMQ24

Any single CpG site

Multiple consecutive CpG sites

– One gene at a time

– Several genes in the same analysis (analyze up to 24 different assays in one run)

Global methylation level

– Estimate the global methylation levels using repetitive elements

Quantification of individual CpG sites

Methylation pattern in RASSF1A in neighboring CpG sites in 4 tumor samples (duplicate runs)

• Methylation levels may vary from site to site

• Pyro Q-CpG detects site variation reproducibly

CpG Methylation Analysis with PyroMarkTMQ24

1. Bisulfite conversion

2. PCR amplification

3. Pyrosequencing

mC mC

C U

T

CmC

U

mC C

25%

75%

Degree of methylation is analyzed as a ”C/T SNP” using the AQ mode in the software

CpG Methylation Analysis

2. PCR amplification

T

CmC

U

1. Bisulfite treatment of denatured DNA

mC mC

C U

3. Analysis

Original sequence:

PCR

GGAmC/CGCTAGmC/CGACTGCTA

GGAmC/UGUTAGmC/UGAUTGUTA

GGAC/TGTTAGC/TGATTGTTA

Bisulfite treatment

AQ mode

• Frequency calculations of variable positions in sequence context including;– Single variable position AG/TC– Multiple variable positions

AG/TCAG/TCAG/T/AC – Di- Tri- or Tetra-allelic mutations GA/C/G/TA

• High resolution of individual sites• Quality assessment of individual sites and

sequence context• SNP analysis (Multiple positions, Di- Tri- or Tetra-

allelic variants)

AQ mode

Quantification of di-, tri-, or tetra-allelic mutations

• Analysis of SNPs in the presence of methylation

• Fast results – typically 15 minutes after PCR to run a typical SNP plate – multiple plates can be run each day

Pyrosequencing delivers quantitative and reproducible data

Pyrosequencing Technology

PPi

ATPTime

Light

PyroMark Q24