Single Nucleotide Polymorphisms (SNPs) are bi-allelic (usually) nucleotide variants that have been found throughout the genome. An example of a hypothetical sequence containing a SNP could look like the following:
CATG [C] TTAAC
CATG [T] TTAAC
There are a variety of technologies for high throughput SNP genotyping in subjects. Although SNPs are less polymorphic (usually only two alleles) than microsatellite repeats, linkage screens can be conducted using a much greater density of SNPs, which more than compensates for the diminished segregation information from individual SNPs.
For projects that require genotyping tens to hundreds of SNPs, mass spectrometry-based platforms from Sequenom provide cost-effective and accurate methods for SNP genotyping. Sequenom genotyping technology is based upon primer extension using allele-specific mixtures of dNTPs and ddNTPs with mass spectrometric analysis of the products. The iPLEX Gold platform on the Sequenom is designed to genotype multiplex pools up to 32 SNPs per well. A custom assay is designed for each pool of SNPs to be genotyped. In general 70-90% of SNPs will be able to have a successful genotyping assay designed.
SNPs are genotyped in a single multiplex reaction using 2 µL of DNA at a concentration of 5-10 ng/µL of genomic DNA. Genotyping of high quality and well purified whole genome amplified samples is also usually
possible. After multiplex PCR amplification, unused nucleotides are eliminated by digestion with shrimp alkaline phosphatase. The extension primers are then added, annealed and extended with an optimized mixture of modified nucleotides. Depending upon the alleles present, the primer extension products will have different masses, and the mixture of primer extension products is analyzed by mass spectrometry. Genotyping software calls each SNP genotype based upon the measured mass of each primer extension product present compared to the calculated mass of all possible extension products for that pool of SNPs.
To begin a project, please contact Jey Moon. Users need to provide a list of variants containing either its rs# or its FASTA sequence with 50 bps up and downstream. When providing FASTA sequences, the reference alleles and variants should be identified in brackets. For example [A/T] or [A/-] where the dash denotes a deletion.
We require 3 µL of DNA at a concentration of 5-10 ng/µL per multiplex pool. 2 µl will be used for genotyping and 1 µl is used to cover pipetting and systematic volume loss. Samples can be submitted in either 384- or 96-well plates. Please note, we only genotype human DNA.
The cost of the oligonucleotides for Sequenom SNP genotyping are for synthesis and storage upto 6 months at our facility for any future projects.
The following is the cost breakdown of our Sequenom SNP genotyping services performed at the SNP Genotyping Facility located at Boston Children’s Hospital. The figures are approximate, based on current reagent, materials and labor costs.
384-well Plate Genotyped: $1,700.00/plate
For example, if your project has 600 samples and 75 SNPs falling into 3 multiplex pools, the cost of the project will be as outlined below:
2 384-well plates x 3 pools : $1,700.00 x 6 plates = $10,200.00
Oligos (75 SNPs): $20.00 x 75 SNPs = $1,500.00
To begin a project, users need to provide a list of variants containing either its rs# or its FASTA sequence with 100 bps up and downstream. When providing FASTA sequences, the reference alleles and variants should be identified in brackets. For example [A/T] or [A/-] where the dash denotes a deletion. We require 3 μL of DNA at a concentration of 5-10 ng/μL per multiplex pool. 2 μl will be used for genotyping and 1 μl is used to cover pipetting and systematic volume loss. Samples can be submitted in either 384- or 96-well plates. Please note, we only genotype human DNA.
Sequenom SNP Genotyping Facility has not received any reviews.
Sequenom SNP Genotyping Facility has not received any endorsements.