How do we perform genotyping?

How do we perform genotyping?

Written by: Nermin Đuzić, M.Sc. in Genetics, Content Specialist

The last time we introduced you to genotyping and its importance. You had the opportunity to learn more about the different applications of genotyping. Now it’s time to shift back to the whole procedure of how DNA genotyping is actually performed. We have explained sampling, DNA extraction, and quality assessment so far.

We will now talk about the steps scientists and lab technicians follow to complete the genotyping assay and obtain genotyping data. As we mentioned in a previous article, there are many different genotyping technologies employed nowadays and these are the most common:

  • 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 the following article, we will give a brief overview of these technologies mentioned above, but now the main focus will be on the technology we rely on. Here we focus on the microarray approach to genotyping.

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 [1]. 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 [2].

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.  This will enable the fragments to attach to hybridization probes on the Affymetrix PMDA (Precision Medicine Diversity Array). Additionally, quality control checks of obtained fragments are performed.
  • Resuspension and hybridization: After the DNA samples are fragmented and precipitated, it is resuspended by mixing and prepared for the hybridization step. The DNA samples are hybridized onto the Affymetrix PMDA array plate for 23 hours.
  • Staining: Following hybridization, the bound target is washed under stringent conditions to remove the non-specific background caused by random ligation events. Each polymorphic nucleotide is queried via a multi-color ligation event carried out on the array surface. After ligation and staining, the plate is imaged on GeneTitan MC Instrument to obtain intensity data from which genotype information is received.
Figure 1: Stepwise illustration of the  genotyping process (Thermofisher Scientific-Axiom Genotyping Solution)

Afterward, 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.

References

  1. Bumgarner, R. (2013). Overview of DNA microarrays: types, applications, and their future. Current protocols in molecular biology, 101(1), 22-1.

  2. Shapero, M., Purdy, M., Kurapati, R., Law, J., Lee, H., … & Bellon, L. SNP genotyping using Affymetrix Axiom® Genotyping Solution, Application Brief, Thermofisher Scientific. 

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