Progress, Results, and Discussion
At this point in the experiment, every step mentioned in the proposal has been performed for Round 1 with the exception of the entire PAGE process due to multiple time commitment issues and mistakes involving previous rounds of selection of other targets. However, even though an entire round has yet to have been performed, it is worth noting that everything has been conducted successfully without any problems. For instance, Gel 1 depicted below is a 3.8% agarose gel with EtBr that was ran to determine the optimal number of cycles for the large PCR.
According to Gel 1, the optimal number of cycles for the target is an estimate between 12 cycles and 15 cycles. It was determined that 14 cycles would be the best choice, considering that after 12 cycles the DNA was under-amplified and after 15 cycles it was slightly over-amplified. Using these results, a large scale PCR was performed for 14 cycles, after which it was precipitated and resuspended at a greater concentration for use in a transcription reaction. Transcription was performed at a temperature of 42 degrees Celsius and quenched with 2X Blue Denaturing Dye to stop the reaction and prepare the sample for a later PAGE gel electrophoresis.
Thus far in the experiment no problems have been encountered during the selection process for HIV-1 Gag p24, but numerous issues were encountered during the subsequent rounds of practice selection of Lysozyme and Maltose Binding Protein. However, these problems will not be discussed in this context considering that they are not related to the current selected target.
Conclusion and Future Work
An entire round of selection has yet to be performed for the selection of HIV-1 Gag p24, but the Round 1 RNA has been successfully selected, reverse transcribed into DNA, amplified for 14 PCR cycles, and transcribed without any major problems. Due to the fact that engineering therapeutics against the formation of functional HIV-1 strain viruses within a human host is the main objective of this experiment, the target and N58 pool RNA were incubated at 37 degrees Celsius, human body temperature, as a means of negative selection. As previously stated, no major issues have appeared in the research process at this point.
Later, the transcribed RNA will be purified via PAGE gel electrophoresis and precipitated at a greater concentration before being analyzed using a form of molecular spectrophotometry to determine the final concentration of the product. This solution will then be used during the subsequent rounds of selection against HIV-1 Gag p24.