biorepository and molecular pathology core banner
slider

iPlexPro Chemistry

iPlexPro Chemistry

The MassArray utilizes Sequenom’s iPlexPro chemistry for SNP genotyping and somatic mutation profiling. The initial step involves PCR amplifying the region spanning the SNP or mutation of interest using forward and reverse primers (80-120 bp amplicons). Subsequent treatment with shrimp alkaline phosphatase (SAP) eliminates unincorporated nucleotides. Extension primers (one for each SNP or mutation) are added to the amplified DNA template along with a thermosequenase enzyme and four different mass-modified dideoxynucleotide terminators. The extension primers are designed to anneal immediately next to the SNP or mutation of interest. During the cycling step, the thermosequenase adds one of the four terminators, which each have a different molecular weight. The reactions are then desalted and 8-12 nL is spotted on a 96 well SpectroCHIP II using the RS1000 Nanodispenser. The chips are fired on the Sequenom MassAnalyzer 4 MALDI-TOF mass spectrometer where a UV laser induces desorption and ionization of the DNA follow by time of flight mass spec detection (heavier ions take longer to travel to the detector). Based upon the initial molecular weight of the extend primer, the MassArray detects which terminator was added and the software assigns a call for each SNP or mutation.

Assays can be multiplexed by increasing the molecular weight of the extend primer. The MassArray is capable of detecting between 15-30 nucleotides with a molecular weight range of 5,000-10,000 Da. For germline SNP detection, a maximum of 30-40 SNPs can be multiplexed into one well. Somatic mutation profiling requires increased sensitivity and only 8-10 mutations can be multiplexed into one well.