The genotyping facility provides high-, medium-, and low-throughput genotyping for projects of all sizes. Because of the unique characteristics of each project, the service is a customized process and it is not possible to quote exact pricing. Therefore we will discuss with the inquiring investigator the proper design and goals of the project. During this meeting the most appropriate technology for the specific project and an estimated cost will be determined. This meeting is free of charge.
The facility offers the following services:
- SNP Genotyping
- Microsatellite Genotyping
Low throughput genotyping for a single SNP for up to 100 samples is performed by sequence analysis (see above).
Medium throughput genotyping (>100 samples and >then a single SNP) can be performed using the following platforms;
High Resolution Melting Curve allelic discrimination: High Resolution Melting Curve (HRM) is a recent development that can greatly extend the utility of traditional DNA melting analysis.
HRM characterizes nucleic acid samples based on their disassociation (melting) behavior. Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes such as SNPs (single nucleotide polymorphisms) can be readily identified and genotyping is accomplished by monitoring the melting of probe-target duplexes post-PCR. This method is suitable for low/medium throughput SNP genotyping, mutation analysis and resequencing.
With HRM low-cost generic dyes are used where previously custom labeled probes such as TaqMan® or fluorescence resonance energy transfer (FRET) probes were required. HRM is thus a simpler and much more cost-effective way to characterize samples.
Taqman Allelic Discrimination
SNP allelic discrimination is the method of choice when a few SNPs are being assayed for a large number of samples. This procedure involves two locus specific primers which surround a SNP and two allele specific probes containing the SNP. The allele specific probes are fluorescently labeled at the 5' end and have a quencher at the 3' end. During PCR, if homology exists between a genomic SNP and an allele specific probe, the 5' fluorescent label is cleaved from the probe (and away from the quencher). This fluorescence is then read by performing endpoint analysis on the ABI 7900HT or Roche Light Cycle 480 instruments. Millions of pre-designed primer/probe sets are now available as wells as customs primer sets.
SNPlexs Genotyping System
The ABI SNPlexTM Genotyping System enables the simultaneous genotyping of up to 48 SNPs against a single biological sample. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects being therefore suitable for a broad range of study designs.
High Throughput genotyping (genome wide analysis or customized arrays).
Affymetrix: The whole range of genotyping arrays from Affymetrix is available. The laboratory has followed the Affymetrix training for the Genome wide SNP array version 5 and 6 and can also support allele calling and Copy Number Variant (CNV) analysis.
- Microsatellite Genotyping:
Microsatellite markers are characterized by differences in repeat length between individuals (alleles), and therefore are analysed by PCR and subsequent separation of the PCR products on automated sequence analysers. Dedicated software is used to determine the alleles of a particular marker (allele calling). We perform genotyping of microsatellite markers (STRs) for genome-wide scans and fine mapping according to yours needs. For genome wide scans for linkage we employ the Applied Biosystems MD-10 marker set consisting of markers with an average spacing of 10 cM.
We accept existing assays just to run them for clients on the capillary sequencer. These should be submitted to the laboratory in clearly labeled 96 well plates, preferably using fluorescent dyes FAM, VIC, NED, PET and LIZ.
We also perform genotyping according to clients needs including optimization of PCRs of microsatellites chosen to suit specific chromosomal regions and density, please enquire for the possibilities with the facility responsible.