Team:HK HCY LFC/Improve
Improvement
In our project, several failure we encountered when assembling and test the feasibility of the DNA nanostructure are listed as followed:
1. Fail in adjusting the concentration of each single strand DNA oligos
2. The three single strand DNA oligos cannot bind to each other
3. Low absorbance of the peroxidase activity assay
In the beginning, the band intensity of oligo 2 and strand 3 are lower than that of oligo 1 (figure 1). After the analysis by a software called ImageJ, the intensity of oligo 1 was doubled that of oligo 2 and 3. Therefore, the amount of oligo 2 and oligo 3 were doubled and the the three oligos were put together for assembly.
Figure 1: The three single strand DNA oligo were loaded into a 12 % polyacrylamide gel and the table on the right shows the content of each lane.
After the assembly of DNA nanostructure using thermocycler, it was found that there was excess banding of oligo 2 and 3 in lane 4, 5 and 7(figure 1). It can be concluded that strand 2 and strand 3 are in excess. We repeated the experiment several times and same result was obtained. Thus, it is deduced that the difference in intensity in figure 1 was due to the ability of the DNA stain to stain the DNA rather than the concentration difference between each oligo. SYBR safe was used to stain the DNA, it preferentially binds to double strand DNA (dsDNA) than single strand DNA (ssDNA) since it is a intercalating dye. [1] Since oligo 1 has a larger size (62 bases) than oligo 2 (36 bases) and oligo 3 (39 bases), oligo 1 has a larger secondary structure than oligo 2 and 3. Therefore, it has a higher chance of intercalating by the dye.
Therefore, the original amount of oligo 1, 2 and 3 were used to test for the interactions between the DNA nanostructure and input DNA oligos. No excess banding of oligo 2 and 3 were observed. (refer to the gel photo in the result page).
Figure 2: Validation of the assembly of DNA nanostructure and the binding between the DNA nanostructure and the target oligos using 12 % polyacrylamide gel electrophoresis. Excess banding of oligo 2 and 3 were observed in lane 4, 5 and 7.
The three single strand DNA oligos cannot bind to each other at the beginning. It was later found out that it was due to lack of cations in the running buffer. The buffer used for assembling the DNA nanostructure is 1X Phosphate-buffered saline (PBS). When the DNA was loaded into the polyacrylamide gel, the concentration of PBS would be diluted by the TBE buffer. Thus, NaCl and KCl were added to the running buffer and the three DNA oligos can bind together.
We prepared some diluted H2O2 and test for each setup one by one. The absorbance at the first few trials were consistent, but it started to drop lower than expected afterwards. The low absorbance was later found out that it was due to the decomposition of H2O2. In order to provide a better result, H2O2 is required to be freshly prepared for each experiment.
References
[1] Haines, A. M., Tobe, S. S., Kobus, H. J., & Linacre, A. (2015). Properties of nucleic acid staining dyes used in gel electrophoresis. Electrophoresis, 36(6), 941-944.
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