Home

Results

According to our methodology, after the DNA extraction, the concentration of the extracted DNA samples and their ratio of purity, regarding the presence of proteins and ethanol is being determined by using Nanodrop. Using the Nucleospin Blood extraction kit results in concentrations ranging from 50 – 100 ng/uL. Herein we represent the concentrations of the 81 examined DNA samples, with the majority being between this range.

As described previously, 81 DNA samples were tested using two methods, on our portable laboratory BentoLab, and on conventional laboratory equipment to double-check the reliability of BentoLab. The genotypes obtained from each method were compared against the KASP assay, referred to as the gold standard method.

Our experiments started by testing the designed primers for the Allele-Specific PCR (AS-PCR) to find the annealing temperature of their function. The check was performed by implementing a Gradient AS-PCR on conventional laboratory equipment for a control sample with a known genotype. According to the image below, the proper annealing temperature is 65℃, where the sample appeared with the correct genotype, homozygous for the ancestral allele.

Figure 1: Representation of the Gradient AS-PCR (62-67℃) of the rs4149056 genotype for a control sample with genotype, homozygous for the ancestral allele T, by using the conventional PCR device. For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp.

Continuing to standardize the AS-PCR protocol, we tested three DNA samples in a conventional benchtop device: IGEM20-020, IGEM20-023, IGEM20-024, applying the annealing temperature 65℃. The obtained results are provided in the photo below. The presented genotypes are correct when compared with the genotypes provided from the gold standard method. Thus, we continue testing the remaining samples implementing this protocol on the conventional laboratory equipment.

Figure 2: Representation of the rs4149056 genotype for three different DNA samples (IGEM20-020, IGEM20-023, IGEM20-024) using the conventional PCR device and applying the standardized protocol for AS-PCR. For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp.

The next step was to standardize the AS-PCR protocol again on BentoLab. We implemented the same protocol with the conventional PCR device, but the results were not satisfying, and we had to modify it.

Figure 3: Representation of the rs4149056 genotype for three different DNA samples (IGEM20-024, IGEM20-023, IGEM20-020) using the BentoLab method with the standardized protocol from the conventional PCR equipment. For the visualization of the results, we used a) the blue LED light of BentoLab (left) and b) the UV device (right). For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp. The results are not satisfying, and the protocols have to be modified.Figure 3: Representation of the rs4149056 genotype for three different DNA samples (IGEM20-024, IGEM20-023, IGEM20-020) using the BentoLab method with the standardized protocol from the conventional PCR equipment. For the visualization of the results, we used a) the blue LED light of BentoLab (left) and b) the UV device (right). For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp. The results are not satisfying, and the protocols have to be modified.

We modify the PCR protocol, the conditions, the electrophoresis protocol respecting the BentoLab’s specifications. More importantly, in the preparation step of the agarose gel, we added the gel stain to visualize the results on the blue LED light of BentoLab because, as we can see in Figure 3, using the Midori gel stain is visible only in UV device. The obtained results respecting three DNA samples (IGEM20-020, IGEM20-023, IGEM20-024 ) are presented in the image below.

Figure 4: Representation of the rs4149056 genotype for three different DNA samples (IGEM20-024, IGEM20-023, IGEM20-020) using the BentoLab method. For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp.

After standardizing the protocols, all the 81 DNA samples were tested on BentoLab. The following table presents the genotypes of the 81 samples as derived from the BentoLab against the gold standard. The ancestral allele is T, and the alternative one is C. N/A indicates that the method results in a different genotype from the gold standard method.

According to the above table, a comparison between the findings obtained from the BentoLab against the given genotypes provided from the gold standard method was conducted. According to that comparison, the BentoLab resulted in 77 out of 81 samples with correctly identified genotypes.

Figure 5: Representation of the number of samples using the BentoLab. True stands for the samples with correct genotype regarding the gold standard method and false for the samples that Bentolab results in a different genotype from the gold standard method.

Below, we attach some indicative photos of the agarose gels after the visualization on the blue LED on BentoLab. For each of the DNA samples, two wells match: for the ancestral allele (‘‘w,’’ referring to T allele) and for the alternative allele (‘‘m,’’ referring to C allele). In the first well, a ladder 100bp was loaded, and the expected molecular weight per PCR product is 535 bp.

The agarose gel electrophoresis' images were used by the dry lab team to train and test the Artificial Intelligencemodel.

Team:Patras/Results

Results

Sponsors

About

Follow Us