To meet the demands of high-throughput SNP analysis in the post-genome era, The Center for Genomic Application has launched a high throughput single nucleotide polymorphism (SNP) genotyping service, using a real-time system that enables rapid SNP genotyping on a SEQUENOM MassARRAY SNP Genotyping platform. With this, the accuracy, analytical prowess and the reliability of mass spectroscopy has been brought to bear genotyping applications on an industrial scale.

High throughput SNP Genotyping :
The principle of the MassARRAY system is the extension of an oligonucleotide probe over a SNP site in a PCR product, with a mixture of deoxynucleotides and dideoxynucleotides, to produce different size products for each allele of a SNP. The extended products are analyzed by SEQUENOM MALDI-TOF mass spectrometry, and the time-of-flight is proportional to mass, permitting precise determination of the size of products generated, which can be converted into genotype information. Because the mass resolution of this method is very high, one can routinely perform multiplexed assays to permit analysis of up to 6 SNPs in one PCR reaction/tube.

Assay Designing :
All assay design is carried out using proprietary Sequenom software, SpectroDESIGNER. As reagent use is independent of the number of PCR products produced in the plex, we aim for the highest plex possible to reduce the per SNP-genotype cost. SpectroDESIGNER produces multiplexed PCR reactions allowing, in our hands, up to six SNPs to be interrogated in a single-well. As well as ensuring no mass clashes, the software also checks for primer-primer, primer-product and product-product interaction to minimize non-specific extensions.

DNA Qantitation by Picogreen :
In addition to the genotyping of individual, amplified DNA, the MassARRAY system can be used to estimate the relative frequencies of alleles in a pool of multiple, amplified DNAs. Prior to amplification and the hME reaction, We do DNA quantitation, by Picogreen. PicoGreen from Molecular Probes, Inc. is a fluorescent nucleic acid stain. Because it is specific for dsDNA, it provides better quantity estimates then a UV spectrophotometer and also has better lower-range quantitation sensitivity

• Rapid sample analysis in less then 5 seconds/sample

• Utilizes SpectroCHIP™ bioarray for proven, high capacity samples launching pad

• Analyze up to 10 SpectroCHIPs without any necessary user intervention

• Fully automated analysis of genotype reactions in MALDI system with automated identification of all samples and controls

• DNA

• Marker/SNPs

1. DNA samples must be sent in 96 well plates. For each DNA plate we receive, we reserve one position to be used as a negative control. You may have duplicate samples on each plate.


Note that multiples of 96 samples are required for Massarray genotyping. 96 samples per plate will be charged regardless of how many customer DNAs per plate exist.


2. Clearly label all your DNA plates. Each DNA plate must have unique ID.
3. All samples will be quantified by Picogreen before proceeding with the genotyping. Regardless, the customer is to submit the expected DNA concentrations.
4. The quantity of DNA required corresponds to at least five times the amount required for the whole genotyping process. 500 ng of DNA will be used for each group of SNPs. 100 ng of DNA will be required for DNA quantification.


Note that the minimum acceptable amount of DNA is 25 ul at a concentration of 100ng/ul.


5. If your DNA is derived from human subjects, please attach the ethics approval with Request Form 1 – General Genotyping Services.
6. If indicated in Request Form 1 – General Genotyping Services, excess DNA will be returned to you once the experiment is done.
7. In naming your samples, please keep the following guidelines in mind: Each sample must have a unique ID. If a sample exists more than once either on the same DNA plate or over multiple plates, use the same ID for all replicates.
8. Please send both a hard and soft copy of the DNA plate layouts in schematic format. Click here for an example schematic DNA plate layout. Name this file in the following manner: Schematic-DNA-plate-layout-YOUR PROJECT NAME.xls.
9. Electronically, please send the DNA plate layouts in list format. Click here to fill out your DNA plate layout in list format using the example as a guide. Place each DNA plate layout in a different file. Name this file in the following manner: DNA-plate-layout-YOUR PROJECT NAME.csv


Please indicate the sample IDs in the Comments column, and whether the samples have been amplified from original DNA in the Tissue Source column.

10. Please contact us for further inquires.

1. The genotyping personnel are available to provide the necessary tools to select your SNPs of interest. If you have regions or genes of interest in mind, our personnel will select SNPs and design primers to suit your needs. In this case, relay this information by clicking here to fill out the request.
2. The genotyping personnel will design primers for your list of SNPs. The following is the required format (click here to fill out the information):



• It would be preferable for the name of the SNP to not exceed 15 characters. Use rs numbers from dbSNP to name your SNPs.
• Send the sequences in TSV format. Each line should be occupied by one SNP sequence.
• Provide at least 150 base pairs of flanking sequence on each side of the SNP.
• Indicate the SNP of interest in square brackets.
• The flanking sequence should be repeat masked, except for the 25 base pairs surrounding the SNP on each side.
• The sequence should include neighbouring SNPs using the IUPAC symbols listed in the table below. (http://www.chem.qmul.ac.uk/iubmb/misc/naseq.html)
• Include the following information after the sequence as shown in the example below and in the following order:



Example sequence format
Please flag any SNPs you absolutely need in your panel, if applicable. Note that if these SNPs do not pass our QC test, they will be removed and we will ask you to replace them.

Note that depending on the sequence provided (the presence of repeats or low complexity sequences), it may be difficult to design appropriate primers for some SNPs. Customers will be notified of the design scores for each SNP and may be asked to replace some SNPs before proceeding with the order.



3. All primers designed for Massarray Genotyping must be ordered through us.
4. Please contact us for further inquires.

• Validation Step

• Production Step

1. Once a set of SNPs is selected, confidence scores indicating how likely each SNP will perform well will be given.
2. Based on this information, the panel of SNPs will be rearranged to remove SNPs with poor confidence scores. Primer will be ordered.
3. All DNAs will be quantified using PicoGreen.
4. If we obtain good quality data, the rest of your plates will be processed as part of the production step.
5. If we obtain poor quality data, we will investigate whether it is due to poor DNA quality or poor synthesis of primers.
6. The first set of genotypes will be obtained from this step and are called ‘Validation Genotypes’.