General Technique
SNP (Single Nucleotide Polymorphism) genotyping is performed using the technology developed by Sequenom (San Diego, CA) called the MassARRAYTM system. The system can be used to detect biallelic or multi-allelic SNPs, as well as one base pair insertion or deletion polymorphisms. The system is completely equipped with software for assay design, oligo check validation, and analysis.
Assays consist of an initial PCR to amplify the region of interest, followed by an allele discrimination step. One to few base-pairs are extended depending on the allele present in the reaction by primer extension chemistry, producing a different size extension product for each allele. Extension products have unique molecular weights that allow the precise identification of the associated genotypes by mass spectrometry.
The MassARRAYTM mass spectrometer analyzes one sample every two seconds. It can analyze up to 10 spectroCHIPs (384-wells each) without any necessary user intervention. The system is capable of analyzing 3,840 multiplexed assays in about two-and-one-half hour period- a daily throughput of more than 7,500 assays.
The reaction starts with the template amplification: 2.5 ng of DNA containing the SNP site of interest is amplified in a 5 μl volume using a 384-microtiter plate format. Artic Shrimp alkaline phosphatase (SAP) is added to samples, which dephosphorilates any residual amplification nucleotides, preventing their future incorporation and interference with the primer extension assay. The MassEXTENDTM primer, DNA polymerase and a cocktail mixture of dNTPs and ddNTPs are added to initiate the hME primer extension reaction. This reaction generates allele specific primer extension products that are generally 1-4 bases longer than the original MassEXTENDTM Primer. A common primer that identifies both alleles is hybridized directly or closely adjacent to the polymorphic site. Nucleotide mixtures are selected to maximize mass differences for all potential MassEXTENDTM products. Appropriate deoxynucleotides are incorporated through the polymorphic site until a single dideoxynucleotide is incorporated and the reaction terminates. Since the termination point and number of nucleotides is sequence specific, the mass of the extension products generated can be used to identify the possible variants without errors. Following the extension reaction, MassEXTENDTM clean resin is added to the reaction to remove extraneous salts that interfere with MALDI-TOF analysis. 15 nl of sample are transferred from the 384-microtiter plate and spotted onto the pad of the 284 SpectroCHIPTM bioarray. The SpectroCHIPTM bioarray is placed into the MALDI-TOF and the mass and correlating genotype is determined in real time with MassARRAYTM RT software.
Assays can be multiplexed to reduce costs and increase throughput.
The system also is equipped with allelotyping software which allows the interpretation of genotype data from pooled samples (Le Hellard et al., 2002). Allele frequencies calculated by a pooling method have an average margin of error of about 4-5 percent in our hands, which is similar to that reported by Bansal et al. (2002). The lower detection limit for minor alleles is usually in the 0.05-0.10 range (Herbon et al., 2003). Although this method is less accurate than typing each sample separate, it is more cost effective and can be useful as a first pass screening or validation for common SNPs.
We have a Caucasian pool available, constructed by adding equal amounts of each of the 100 Caucasian DNAs (CAU100, Coriell) in a single tube. Prior to pooling, the DNA concentration of each DNA was measured using the PicoGreen dsDNA quantitation reagent (Molecular probes) and adjusted. This method provides a very accurate estimate of DNA concentration of each sample which is needed for proper pooling. Although our pool has only 100 subjects, bigger pool sizes have no significant effect on frequency estimations or repeatability.
Purpose/use/application of the technique:
SNPs are caused by a single base change in the genome sequence and they are the most abundant DNA variation in humans (on average 1/ 300 bases). They are found both in genes and non-coding sequences and some of them are expected to be the cause for common diseases.
Because SNPs are so abundant, they can be used as genetic markers to detect association between a particular genomic region and the disease. SNPs in coding sequences and promoter regions may be directly responsible for a disease phenotype and can be directly tested for correlation with the disease.
Example(s) of projects at ISB that use this technique:
Analysis of Toll-like receptor polymorphisms and their role in infectious diseases.
Association mapping of prostate cancer susceptibility genes in candidate regions identified by linkage mapping.
Representative publication(s):
Hawn TR, Verbon A, Janer M, Zhao LP, Beutler B, Aderem A. Toll-like receptor 4 polymorphisms are associated with resistance to Legionnaires' disease. Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2487-9.
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