Sequenom MassARRAY®

The Sequenom MassARRAY® system is DNA analysis platform that efficiently and precisely measures the amount of genetic target material and variations therein.

DNA Methylation Analysis

The MassARRAY® system can measure individual methylation ratios for CpGs within a target sequence. Relative methylation ratios can be assessed in a range between 10-90% with a standard deviation of 5%. The system allows for flexible experimental design and is scalable for the analysis of a few or several hundred regions over multiple samples. Long reads up to 600bp in one reaction enables discovery of differential methylation within large promoter regions.

Choose from pre-validated assays in the EpiTYPER panels or design custom assays with EpiDesigner (epidesigner.com)

How it works:

• Bisulfite treatment followed by PCR with a T7 promoter tag
• SAP treatment cleans up left over single stranded DNA
• in vitro transcription with T7 Polymerase
• Base-specific RNA cleavage in which the resulting cleavage pattern depends on the presence of methylated cytosine in the original genomic DNA
• Resin clean up to get rid of extra salt
• Nanodispensing on SpectroChip
• Data acquisition with MALDI-TOF mass spectrometer provides quantitative analysis of methylation ratios

SNP Genotyping
The MassARRAY® iPLEX Gold is the leading technology for SNP genotyping for sub-whole genome study applications. It is used for fine mapping and validation studies. Assays can be multiplexed from 2-36 SNPs per individual reaction. The assay design is automated and straight forward.

How it works:

• PCR amplifies ~100bp fragment containing SNP of interest
• SAP treatment cleans up left over primers and dNTPs
• Single-base extension reaction resulting in fragments of different masses
• Resin clean up to get rid of extra salt
• Nanodispensing on SpectroChip
• Data acquisition with MALDI-TOF mass spectrometer provides detection and ratio analysis

What is MALDI-TOF?

Matrix-assisted laser desorption/ionization – time of flight mass spectrometry.

Sequenom’s SpectroChip is coated with a matrix which allows crystallization of the PCR product on its surface. A laser is then fired at the crystal which ionizes the molecules. These ions fly through a vacuum tube to an ion detector based on their mass. Smaller molecules travel faster than larger ones. Time of flight measures the difference in time be molecules hit the detector and the software calculates the mass of the fragments. MALDI-TOF can resolve mass differences of 16 Daltons.

Experiment Guidelines

DNA Methylation

After using the EpiTYPER panels or EpiDesigner, provide the database output which includes the Amplicon Name, Description, LPL, RPL, and the target sequence. Also provide the primers necessary for the amplification PCR. If you want to use your previously designed primers, you must provide these primers with the T7 promoter region before they can be used on the MassARRAY system.

The DNA samples work best if they are eluted in water, RNase free or HPLC water is fine.  The instrument is very sensitive to salt, so avoid TE buffer. Each reaction requires 1uL of 10ng/uL DNA. The DNA can be submitted in 96-well or 384-well plates with at least 2 open wells for negative controls. It is best if 260/280 is between 1.7 and 2.0.

SNP Genotyping

This instrument can perform up to a 36-plex, but in our experience the actual plex will contain between 25 and 30 loci.  To get the largest plex possible, it would be best to provide 40-50 loci with specific notes about their importance.  As for any multi-plex, the number of loci it contains depends on primer interactions, so there is no guarantee that we will be able to design a 36-plex.  Provide the SNP ID (rs# is fine) and the sequence. Within the sequence the SNP should be denoted in brackets (i.e. [G/A]), with 100-200 bases of flanking sequence on either side.  Tri-allelic SNPs are also detectable.  If there are other known polymorphisms within the sequence please denote them as X so that we can design the primers around them.  There cannot be polymorphisms bracketing the SNP of interest within 20bp.  If there is a polymorphism on one side, we can design the primer on the antisense strand.

The DNA samples work best if they are eluted in water, RNase free or HPLC water is fine.  The instrument is very sensitive to salt, so avoid TE buffer. Each reaction requires 1uL of 10ng/uL DNA. The DNA can be submitted in 96-well or 384-well plates with at least 2 open wells for negative controls. It is best if 260/280 is between 1.7 and 2.0.

Price

DNA Methylation

The cost to run one 384-well plate is $1651.00, not including the price of primers. Primers are unlabeled and usually 20-30 bp in length.

SNP Genotyping

The cost to one 384-well plate is $1408.00, not including the price of primers. There are 3 primers per SNP, two for amplification and one for the extension reaction. They are unlabeled and usually 15-25 bp in length.