Team:TUDelft/Project/Results
ADOPE
The aim of ADOPE is to develop a complete method for gene doping detection. The final method consists of four main steps: sample preparation, prescreening, targeted library preparation and DNA sequencing and analysis.
1. Sample Preparation
In the sample preparation module we developed a workflow to extract the target DNA from blood. As a proof of concept, we verified the extraction of the cell free DNA (cfDNA) from bovine serum. Finally, we successfully extracted gene doping DNA that we spiked to the serum, guided by the concentration value generated by the kinetic model.
Design:
We modeled the process of infection and degradation of gene doping DNA in blood to determine its concentration overtime. We use this model to provide approximate sensitivity requirement for DNA extraction.
Results and interpretation:
Based on our model, we determined microdosing was the best doping method for doping athletes, as it avoids high EPO fluctuation, and hence avoids detection through the biological passport. Figure 2 displays the predicted amount of doping DNA fragments in the blood over time as a result of intramuscular (IM) injection every 20 days. Based on this model, we predicted that there will be 40,000 to 50,000 fragments of DNA per mL blood. We based the sensitivity requirement of DNA extraction on this value.
Design: We extracted cfDNA from bovine serum and confirmed successful extraction with Qubit dsDNA HS assay and nested PCR.
Results and interpretation:
We performed cfDNA isolation from the serum using QIAamp DNA Blood Mini Kit. To verify extraction, we quantified cfDNA on the serum using Qubit. We obtained 0.394+/- 0.0149 ng/uL of cfDNA after extraction. To verify the extraction further, we performed nested PCR on albumin gene. Albumin gene is often used for verification of the presence of genomic mammalian DNA and was recommended by Sanquin. The nested albumin PCR consists of a first round of PCR using primers 091 and 092, amplifying an internal fragment of the bovine albumin gene of 229 bp. We deliberately chose to amplify a short fragment since cfDNA in blood is fragmented. The product of the first round of PCR was used as the template for the second round. We used internal primers 066 and 067 to amplify a 150 bp internal fragment of the first PCR product.The product of the first and second round of PCR are shown on Figure 3. The bands on Figure xx lane 2-7 confirmed the success of the extraction.
Design: We spiked samples with artificial gene doping DNA and confirmed its extraction with PCR.
Results and interpretation:
Guided by our kinetic model, we spiked samples with 2x10^8 fragments/ml of either linear or plasmid EPO cDNA. We performed extraction, followed by PCR to verify it. Two EPO internal primer combinations were used. For the primer set 072 & 073 the expected band size was 297 bp and for the primer set 074 & 075 the expected band size was 140 bp. Figure xxx confirmed the successful extraction of gene doping EPO DNA. The difference in intensity between the linear DNA spiked (sample 3 & 4) and the plasmid spiked (sample 5 & 6) indicates that the QIAamp DNA Blood Mini Kit has a lower extraction efficiency for plasmids, since the same amount of fragments were spiked.
Conclusion
We showed successful extraction of artificial gene doping DNA at concentration predicted by our model using the QIAamp DNA Blood Mini Kit. Further extraction optimization is advisable to maximize extraction efficiency.
2. Prescreening
In this module, we developed a high throughput screening method to detect gene doping. The method is based on the concept of color changes induced by d-AuNPs aggregation. In this module, we first analysed the stability of d-AuNPs upon introduction of NaCl with and without single stranded DNA probe. Finally, we were able to visually differentiate between samples where target DNA is presence or absence.
Design:
We generated batches of dextrin-capped gold nanoparticles and tested their aggregation upon NaCl introduction to determine the optimal NaCl concentration.
Results and interpretation:
Upon increasing NaCl concentration, d-AuNPs destabilize and aggregate, changing the color of the solution from red to purple. We scanned the visual light spectra from 450nm to 650nm for a range of 0mM to 333mM NaCl. The important absorbance wavelengths are 520nm (red color) and 620 nm (purple color). We calculated the ratio of 620nm and 520nm to quantify the expect of color changes (red to purple) (figure 5). We observed an increase in the 620/520 with an increase in NaCl concentration, indicating a color change from red to purple.
Design:
We tested the effect of single stranded DNA probe (ssDNAp) on d-AuNPs aggregation.
Results and interpretation:
ssDNAp are known to stabilize d-AuNPs from aggregating (Baetsen-Young, et al, 2018). We incubated the d-AuNP's with and without a ssDNAp and compared the d-AuNP aggregation at a range of NaCl concentrations. The stabilization effect can be observed on figure 6. The addition of ssDNAp causes a lower 620/520 ratio shift with increasing NaCl concentration.
To further evaluate the influence of ssDNAp concentration on the d-AuNPs stabilization, we measured the stability at different NaCl concentrations with varying ssDNAp concentrations. Increasing ssDNAp concentration stabilizes the d-AuNPs, reducing the aggregation and changing the color of the solution from red to purple.
Design:
We incubated DNA with targeting ssDNAp or non-targeting ssDNAp to show the visual difference between the two.
Results and interpretation:
The ssDNAp was incubated with the corresponding target gene doping DNA to let the ssDNAp anneal and form the secondary structure. Subsequently, the d-AuNPs were added and their aggregation was analysed. The ssDNAp targeting EPO was compared with a ssDNAp that is not targeting EPO. The on-target ssDNAp stabilized the d-AuNPs as the ratio 620/520 wavelength was lower compared to off-target ssDNAp (figure 8).
After showing the functionality of the d-AuNPs to detect target DNA, we aimed to determine the sensitivity by using variations of target EPO DNA concentrations while keeping the total amount of DNA constant. We tested concentrations ranging from 1 pM to 1 nM comparing on- and off-target primers and determined our lower limit of detection is 1nM. To achieve comparable results to Baetsen-Young et al. 2018 (29fM) further optimization are required.
Additionally, we evaluated the influence of background DNA present in the sample. We used 38 ng/µL background DNA, equal to the expected cfDNA extraced during sample preparation. We were able to show that the presence of background DNA does not affect the sensitivity of the prescreening method. Further sensitivity evaluation are required before implementing the prescreening method.
Conclusion
The functionality of the prescreen was proven. We were able to visually detect the presence of target EPO DNA. However, to implement this prescreening, protocol optimization is required to reach a lower detection limit of at least 9fM, based on data from our model. Thus, if the results of Baetsen-Young et al. 2018 can be reproduced, the detection limit can be lowered to 2.94fM.
3. Fusion Protein - dxCas9-Tn5
In order to achieve targeted sequencing, we constructed a fusion protein by linking dxCas9 with Tn5 to be used in library preparation for sequencing platform. We constructed the strain that expressed our fusion protein, successfully purified them and tested their functionality in vivo and in vitro. We were able to show targeted integration by our fusion protein in vitro and sequence verified the integration products.
Design:
We constructed a fusion protein by linking dxCas9 cDNA to Tn5 cDNA with a linker coding for glycine helical peptide.
Results and interpretation:
The sequences encoding the dxCas9 (3.7) and Tn5 were obtained via Addgene with plasmid numbers #60240 from Picelli et al. (2014) and #108383 from Liu et al (2018) respectively. First, we introduced a linker coding for glycine helical peptide to Tn5 cDNA and cloned it to pACYCDuet. We screened colonies with the correct insert by colony PCR, followed by visualization of amplified insert on a 0.8% TAE agarose gel (figure 9). Plasmid was isolated from colony 8 and sequence verified.
Next, we introduce dxCas9 into pACYCDuet-1_Lin-Tn5. We screened colonies with the correct insert by colony PCR, followed by visualization of amplified insert on a 0.8% TAE agarose gel (figure 10, 11, 12). After plasmid isolation from positive colony 2, we sequence verified plasmid pACYCDuet-1_dxCas9-Lin-Tn5 (figure 10).
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4. Targeted Sequencing with dxCas9-Tn5
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5. References