State-of-the-Art Technology Combined with Years of Genotyping Experience
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.
The principle and processing using the MALDI-TOF MS hME is same as that is for the MALDI-TOF MS iPLEX.
The only difference being that MALDI-TOF MS hME uses traditional dideoxy nucleotides in the mass extension reaction. This could result in one or two base incorporation to discriminate between the two alleles. This also results in non-symmetric signals for the two alleles making it difficult to achieve higher plex levels. This method can be utilized and is sometimes preferred to iPLEX in determining repeat polymorphisms such as UGT1A1. BioServe has designed and validated this assay to analyze several sample sets.
MassARRAY SNP Analysis
Homozygous low mass allele
Homozygous high mass allele