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SNP - Sequenom
Sequenom SNP genotyping is based on primer-extension reaction that generates allele-specific products with distinct masses. The detection of these products is performed using MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time-Of-Flight) mass spectrometry. This method is applied for custom genotyping of interesting SNPs and uses samples placed in 384-well plates. The main application of Sequenom genotyping is to genotype large number of samples (several hundreds to thousands) and a medium number of SNPs (tens to hundreds).
Following Sequenom applications are available at FIMM Technology Centre:
- Sequenom iPLEX Gold chemistry
iPLEX Gold chemistry is currently the mainly used method. In this method up to 36 SNPs can be simultaneously assayed in one multiplex with standardized conditions.
- Sequenom hME chemistry
This is an older method where sufficient mass differences between the extension products are achieved using 2-3 bp extension products in conjunction with single base extension products. The multiplexing capacity of this method is relatively low (up to 7) but in projects involving only a few SNPs this might still be a reasonable choice.
Our service includes all steps from the assay design through to genotyping and quality control. Assay design is based on the list of SNPs of interest provided by the customer. Information about the importance of each SNP can also be taken into account and multiple design cycles are typically run until the customer is satisfied with the end result. Alternatively, customer can only list the genes of interest and the SNP selection (tagSNPs, non-synonymous coding SNPs, SNPs with known regulatory function) is performed in collaboration with us.
For other applications suitable for the Seqeunom platform, including DNA Methylation, please contact us for information about current availability.
Sequenom genotyping workflow consists of several steps:
SNP selection
Most SNPs (including tri-allelic and small insertion-deletion polymorphisms) are suitable for Sequenom genotyping. However, there are certain limitations that have to be taken into account:
- Unique PCR primers are needed for good quality amplification, thus SNPs located in a duplicated genomic region or near extensive repetitive sequence elements will fail
- Other SNPs should not map to the nearby (~ 20 bp from the targeted SNP) area at least on one side of the SNP
- If the list of SNPs of interest includes SNPs that do not have rs-numbers the customer should send us the sequence (+/- 200 bp) surrounding the variation.
FIMM Supporting equipment for Sequenom applications:
- Tecan Robotic Microplate Processor 150
- Beckman Multimek Automated 96-channel Pipettor
- Sequenom MassARRAY Nanodispenser RS-1000
- MassARRAY Compact System – Bench top MALDI-TOF mass spectrometer