We utilize the MassARRAY iPLEX™ platform by Sequenom for high-throughput SNP analysis. This process combines the technologies of mass spectrometry, polymerase chain reaction (PCR), and primer extension to determine each allele.
The first step in this process is to perform PCR to amplify the region surrounding the SNP. Second step is to treat the PCR reactions with shrimp alkaline phosphatase to dephosphorylate unincorporated nucleotides. In the third step a single base extension (SBE) reaction is performed using thermo sequenase to incorporate mass modified dideoxy nucleotides. This post-PCR primer extension reaction generates small DNA products that have unique mass value based on each allele. These mass extension products are spotted on a chip consisting of matrix that will help in the ionization of the DNA products when exposed to high intensity laser. The time of flight of these ionized products will depend on the mass of each allele that will be measured by the mass spectrometer.
The entire process is completely automated including assay development, PCR setup, post-PCR treatment, nanoliter transfer of extension products to chips, serial reading of chip positions in the mass spectrometer, and final analytical interpretation. The mass modified SBE nucleotides allow for use of highly stringent calling thresholds and performance at high plex levels. This platform utilizes minimal DNA, 5-10 ng of DNA per set of multiplexed assays and provides call rates of greater than 95% on high quality samples that are accurately quantitated.
Quality control (QC) analysis includes typing internal positive control samples, no template controls and 10% replicate samples. Genotyping plates are reviewed for results from positive / negative / DNA control wells that are organized in specific patterns to assist in the QC process and to ensure correct plate orientations during processing and data review.