Over the past two decades, DNA genotyping technologies have been continuously improving. Projects once thought impossible, now succeed in achieving accurate genetic information in a remarkably short time frame.

High-throughput technology refers to the rapid analysis of a large quantity of data points. However, while the total number of resulting data points may be the same, genotyping a small number of samples for many genetic markers (hundreds or thousands) is an entirely different task compared to genotyping many samples (thousands) for a small number of markers.

There are several technologies and platforms suitable for genotyping for plant breeding and production quality purposes; each has its advantages and disadvantages. To select the most suitable technology, Gene-G has examined the available solutions against industry needs, addressing the challenge of identifying which technology best meets customer requirements.

There are two main methods for genotyping with Real-Time PCR:

1. Dual Probe TaqMan: real-time PCR reaction with two additional oligos (probes) marked with two different fluorophores. As the PCR amplification process continues, the fluorescence signal increases, allowing for the distinction between desired and undesired traits.
2. KASP™ (by LGC Ltd.): patented PCR-based genotyping method that is also fluorescence-based (like the TaqMan), but with a different chemistry. The fluorophores are in the assay mix already, and thus there is no need for the addition of specific fluorescent probes.

Between the two PCR technologies, the final cost per data point is the same, but the resource cost breakdown is different. In KASP, the mix is more expensive due to the cost of fluorophores, while in TaqMan, the probes are more expensive. This means that in marker development, while testing different SNPs and probes, TaqMan is more expensive than KASP, but in routine marker scans, there is no difference in cost per data-point.

KASP is only an end-point PCR, while TaqMan can also be conducted as real-time PCR. This feature raises the accuracy of the data obtained. This is critically important in seed quality assurance processes (variety and parental line purity) and in many other industries, such as medicine.

Real-time PCR technologies are better suited for genotyping projects of many samples on a small number of markers. To increase testing capacity, several automated platforms for real-time PCR are available, such as Fluidigm (by Standard BioTools) and Intelliqube (By Biosearch Technologies).

Hybridization Microarray: hundreds of thousands of probes are arrayed on a small chip, allowing for many SNPs to be genotyped simultaneously. This method is suitable for genotyping a small number of samples for many genetic markers. Because this method is very sensitive, clean DNA of high quality is required, which increases the cost. This method is a bit too expensive for common commercial companies in the industry and is therefore more commonly used in academia.

The aforementioned technologies rely on different sequence hybridization; however, when the alleles differ in amplicon length (such as InDels and SSRs), it is also possible to distinguish between them by length. Following a conventional PCR reaction, the PCR amplicons are separated by size using capillary electrophoresis. This technology can differentiate between multiple alleles within a population in a single assay. Unfortunately, it is difficult to upscale this method for projects with many samples across many markers.

Another technology differentiates between the alleles by their mass. When two alleles are the same length but have different masses, such as SNP, size separation is not possible, whereas mass separation is. This small difference can be distinguished according to the physical principle “Time of Flight”. The MassArray technology (by Agena Bioscience) can analyze up to 60 markers per sample across 384 samples in a single run, resulting in 23,040 data points.

This Agena technology is effective for genotyping thousands of samples on a medium number of markers.

After careful consideration, Gene-G has decided to implement two complementary technologies: TaqMan and MassArray.

• TaqMan technology is better suited for marker assisted selection (MAS)
• MassArray technology is most effective for ensuring genomic purity and uniformity among populations, particularly in variety production quality control
• Both technologies – TaqMan and MassArray are essential for marker-assisted backcrossing (MABC)

If you are considering various technological alternatives for your marker operations, Gene-G provides consultancy services to help you make the right decision.