Written by: Nermin Đuzić, M.Sc. in Genetics, Content Specialist
Our previous article provided a brief overview of how Affymetrix genotyping technology is performed as we rely on it. In this article, we will briefly discuss other main genotyping technologies widely used nowadays for various purposes of analyzing DNA.
Genotyping technology has been subjected to enormous and explosive advancement during the last few decades. These mainly include the availability of arrays that may accommodate the analysis of millions of SNPs, and increased cost savings, that were even unimaginable several decades before. All these have resulted in an explosion of genome-wide association studies and the identification of many new SNPs responsible for the diseases .
The most prevalent genotyping technologies used nowadays are those based on medium and high throughput screening approaches such as:
- SNP microarrays (Illumina and Affymetrix being the best known)
- TaqMan SNP genotyping
- MassARRAY SNP genotyping
- High-throughput DNA sequencing.
SNP microarray is also known as a DNA microarray, 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 many genes, and genotyping .
One of the most widely used and popular genotyping microarray technologies is the Affymetrix GeneChip Array, which is cost-effective and enables the automation of sample preparation and other genotyping steps.
It is based on allelic discrimination by direct hybridization of genomic DNA to arrays containing locus- and allele-specific oligonucleotides. We have already explained how we use Affymetrix Array technology in our labs. If you missed it, you can read it in detail here. Therefore, we will shift to another SNP microarray technology- Illumina.
Illumina genotyping assays
Illumina genotyping technology comes into several forms, among which we can mention Illumina’s Infinium Beadchip assay and Illumina Goldengate assay.
Illumina’s Infinium assays can be used for up to 1 million multiplexed assays. It includes a whole-genome amplification step and hybridization to bead arrays. The bead arrays have 50 base pairs-long capture probes.
Also, beads contain locus-specific probes, and allelic discrimination occurs through primer extension reactions. Characteristics of Infinium assays are high pass rate and accuracy 
On the other hand, the Goldengate Illumina assay is suitable for a smaller number of SNPs, as it covers the spectrum of 96-1536 SNPs and allows array customization. It also relies on bead chip technology from Illumina.
The technique is based on PCR amplification and extension of primers, followed by ligation and amplification with fluorescently labeled primers. This system possesses a high-resolution scanner and computer workstation to detect fluorescent beads and decode the information used for genotype calls. This assay has a high pass rate and accuracy. [4, 5]
TaqMan SNP assay
Besides Affymetrix and Illumina, another often used genotyping approach is TaqMan SNP assay, which is based on a nuclease assay on a 5’ end of DNA. Nuclease is an enzyme that cleaves the chains of nucleotides into smaller units. TaqMan uses nucleases at the 5’ end of DNA chains. It is called TaqMan because it uses Taq polymerase, a thermostable DNA polymerase enzyme isolated from the bacterium Thermus aquaticus .
The assay takes place in a single tube or well and requires a real-time PCR machine to detect fluorescence. Advantages of this method are that it requires only one simple reaction to set up, and a huge number of prevalidated assays are available. However, a major advantage is that it allows you to customize your own array for a specific problem. Also, the whole system can be completed within 8 hours, and up to 98 thousand genotypes can be produced per day per system [1, 7].
MassARRAY SNP genotyping
Another technology that uses PCR is MassARRAY SNP genotyping. This technology uses PCR to amplify DNA sequence across the SNP site and then uses a single extension primer to amplify PCR products. Afterward, PCR products are analyzed, and SNPs are classified according to the molecular weight differences between bases. Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry (MS) is used to determine the molecular weights.
Also, it is possible to calculate the allele frequencies of SNP sites. This method allows multiple SNPs typing within a single reaction. Also, it is rapid and accurate. MassARRAY SNP genotyping has wide applications in biological and medical sciences, agricultural research, etc..
Finally, we have another high-throughput method, which is not genotyping per se. It is DNA sequencing, and there are several generations of sequencing technologies with next-generation sequencing currently being the most popular. However, there are also third-generation sequencing technologies such as Oxford Nanopore.
By definition, DNA sequencing determines the order of nucleotide bases within a DNA molecule. How is sequencing different from genotyping, you may ask.
Well, to understand the difference between these two, imagine a book. Imagine nucleotide bases (letters A, C, G, T) that build your genetic code are like words on the book page. Now we can read that book by either sequencing or genotyping approach. The difference is that genotyping is like reading particular and scattered words on a page, while sequencing reads the whole content or each word.
Genotyping is useful if we know what we are looking for, as it is a great choice for finding something we know today. However, this “targeted-word” search approach may sometimes cause ambiguities and incompleteness. For example, reading the word “knife” does not give you a clue whether the book is about cooking or it tells some famous murder mystery.
While genotyping provides only a small and targeted amount of data, sequencing offers more complete and comprehensive information. Next-generation sequencing allows us to see beyond the commonly known genetic variations. It enables scientists to create more variations unique to a person. Finally, it helps us understand how DNA underpins our health and lifestyle.
A big advantage of NGS is that the whole human genome can be sequenced within a day. Numerous NGS platforms use different sequencing technologies, but all of them are based on parallel sequencing of millions of small DNA fragments. Bioinformatics analyses are employed to piece together these fragments by mapping individual reads to the human reference genome .
After learning more about genotyping and sequencing technologies, it is time to move forward and explain the next steps towards the final report generation. More on that in the next articles. Stay tuned!
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- Chien, A., Edgar, D. B., & Trela, J. M. (1976). Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. Journal of bacteriology, 127(3), 1550-1557.
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- Behjati, S., & Tarpey, P. S. (2013). What is next-generation sequencing?. Archives of Disease in Childhood-Education and Practice, 98(6), 236-238.