Single Nucleotide Polymorphism Detection with the MassARRAY system from Agena
Since its introduction in 2000, the MassARRAY system (previously named Sequenom) has been widely adopted for a variety of genomic applications. Research fields like routine genetic analysis of SNPs, linkage studies and validation of genome-wide association studies took advantage of this technology.
- MassArray, a technology able to analyze hundreds of mutations on a hundred of samples
- Main characteristics of the system
- The first step: to design our experiment
- The magic effect of iPLEX chemistry. How is working?
- Is the acquisition of the raw data and its interpretation complicated?
- Assays by Agena: main applications
MassArray, a technology able to analyze hundreds of mutations on a hundred of samples
Agena allows us to analyze a variety of biomarkers from a full spectrum of biological samples. This starting material can be: blood, plasma, buccal swabs, and even compromised formalin-fixed, paraffin-embedded (FFPE) samples. With as little as 5 ng of DNA we can start our experiment.
SNP genotyping on the MassARRAY System combines the robust multiplexed primer extension chemistry with highly sensitive mass spectrometry. This combination gives precision, fast, and cost-effective analysis from hundreds of genotypes per day.
Main characteristics of the system
Next generation sequencing (NGS) and in general all genome association studies have stirred up the field of Genetics, facilitating the discovery of new genetic markers and contributing significantly to personalized medicine.
However, these candidate markers require complementary studies to validate them as a molecular diagnostic marker. MassARRAY technology is focused to provide solutions fastly and concisely. That is why Assays by Agena allow us to analyze a combination of SNPs on 96- or 384-wells.
Furthermore, this technology allows us to analyze at least 40 SNPs per well.
Another of the main advantages of the use of MassARRAY technology is its workflow. The results report is obtained in less than one working day and with only 30 minutes of pipetting. Here we will explain you more in details.
The first step: to design our experiment
The first step to perform our experiment is to design it. Using Assay Design Suite (ADS) software from Agena, we can design the PCR and iPLEX extension primers for each SNP or somatic mutation. We only need to provide to the software the accession number on FASTA format. Depending upon the complexity of the assay, a single multiplex reaction can contain up to 40 markers. All oligos for PCR amplification are ordered unmodified, with standard purification, minimizing assay setupo costs. Alternatively, predesigned panels from Agena contains the necessary premixed primers and reagents.
In this webinar we are showing you how easy is to design your own experiment using Assay Design Suite software from Agena.
The magic effect of iPLEX chemistry. How is working?
Once the PCR and extension primers have been designed and acquired, genomic DNA or cDNA is amplified in 96 or 384-well microtiter plates using reagents supplied in the iPLEX reagent kits. PCR conditions like: reaction volume or conditions of the PCR program are determined by the kit.
After PCR, excess nucleotides are dephosphorylated by shrimp alkaline phosphatase (SAP). This is follwed by the iPLEX single base extension reaction in which a mix of oligonucleotide extension primers, designed to anneal to the amplified DNA fragments, is added together with an extension enzyme and mass-modified dideoxynucleotide terminators.
Two iPLEX reagent options are available. iPLEX Gold is intended for standard genotyping, while iPLEX Pro is available for more demanding assays that require higher performance and sensitivity, such as in diagnostic and clinical applications.
Using iPLEX reagents you could amplify, extend and detect your SNPs!
Is the acquisition of the raw data and its interpretation complicated?
The answer is no. Once the SpectroCHIP array is loaded into the MassARRAY Analyzer, where the analyte crystals are irradiated and ionized in order that the charged molecules accelerate into a detector. Separation occurs by time-of-flight, which is proportional to the mass of the individual molecules.
The entire process takes less than 50 minutes to analyze 384 samples. In this picture, we can show you how looks a complete spectrum for an experiment of 36-plex assay. The highlighted peaks indicate 24 Da separation in heterozygous alleles (B) of the SNP in question compared to the single peak homozygous genotype (C).
Typer software automatically generates reports that identify the SNP alleles (homozygous or heterozygous) in each sample.
Assays by Agena: main applications
The services offered by MassARRAY technology are divided into three fields of study: oncology and liquid biopsy, pharmacogenetics and sample identification.
The collection of panels dedicated to oncology, highly multiplexed, allow to outline a wide variety of samples suspected of presenting cancer. These panels can be applied to a wide variety of biological samples. In this range of products, we find the panels dedicated to liquid biopsy. If you want more information, you can pick up the post in which we talk about liquid biopsy and Ultraseek panels.
The second focus of interest of the MassARRAY platform is pharmacogenetics. This field of biomedicine, allows to evaluate how a patient respond to a treatment. The combination of this platform together with the knowledge of environmental factors and the heterogeneity of the disease, allows to guide the appropriate therapeutic treatment. Try to remember what we explain you about pharmacogenetics visiting this post.
Finally, the sample identification, is another of the main fields of Agena bioscience. The panel SampleID is able to multiplex 52 different SNPs per well and allows to identify problems related to the management of biological samples.
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