Genotyping is the process of analysing DNA sequences to determine genetic variation. At BioCertica, we use the SNP microarray to analyse your individual genetic makeup.
So far, we have explained sampling, DNA extraction, and quality assessment. We have also introduced genotyping and its importance, in which we spoke about the different applications of genotyping and the many different genotyping technologies employed with the most common being
- SNP microarrays (the best known are ones provided by Illumina and Affymetrix)
- TaqMan SNP genotyping
- MassARRAY SNP genotyping
- and SNP screening by high-throughput.
In this article, we will focus on the procedure of BioCertica's genotyping technology: the microarray.
A DNA microarray is also known as a microchip or biochip and represents a collection of microscopic DNA spots on a solid surface. It is used to measure gene expression, allow the simultaneous expression analysis of a large number of genes, and for genotyping . If you want to know more about DNA microarrays, we suggest watching this video and seeing how it is practically done.
At Biocertica, we use Affymetrix Axiom genotyping technology and use PMDA Plus Array on the GeneTitan machine. This technology is cost-effective and enables complete automation of sample preparation and other steps, briefly explained below. Also, this assay contains 96 individual arrays and allows genotyping of up to 800 000 SNPs, with a throughput of ~200 samples per month .
The whole process consists of the following steps (depicted in Figure 1 below):
- DNA amplification: Once DNA is extracted and passes quality control of DNA, we can proceed with DNA amplification. The amplification step aims to enrich the DNA for the successful completion of the next steps. A total of 100 ng of genomic DNA is amplified.
- Fragmentation and precipitation: After DNA amplification, the DNA is fragmented and precipitated. During these steps, the aim is to break DNA into shorter fragments of 25-125 base pairs and to concentrate the DNA. The fragmentation will enable the fragments to attach to the probes of the Affymetrix PMDA plate (Precision Medicine Diversity Array) in the following step.
- Resuspension, QC and hybridization: After the DNA samples are fragmented and precipitated, it is resuspended and prepared for the hybridization step. A quality control step is also implemented to check the amplification and fragmentation before hybridization. The DNA samples are then hybridized onto the Affymetrix PMDA array plate for 23 hours to allow your DNA to bind to the oligonucleotide DNA on the probe.
- Staining: Following hybridization, the plate with the bound DNA is washed under stringent conditions to remove the remaining unbound product. Then, each polymorphic nucleotide that has bound to the oligonucleotide DNA on the probe is queried via a multi-colour ligation event carried out on the array surface. After ligation and staining, the plate is imaged on the GeneTitan MC Instrument to obtain intensity data from which genotype information is received.
Afterwards, the results are ready to analyze and generate the report. How? We will explain this in one of the following articles.
In the meantime, you may wish to watch the entire genotyping procedure or look for additional information. If so, you can refer to this video.
Written by: Nermin Đuzić, M.Sc. in Genetics, Content Specialist
Bumgarner, R. (2013). Overview of DNA microarrays: types, applications, and their future. Current protocols in molecular biology, 101(1), 22-1.
Shapero, M., Purdy, M., Kurapati, R., Law, J., Lee, H., … & Bellon, L. SNP genotyping using Affymetrix Axiom® Genotyping Solution, Application Brief, Thermofisher Scientific.