Challenges:
The main challenge of this system for amplification is that the reaction requires special chemistry to work properly and some cell components interfere with PCR. Since all steps happen on one chip, all the fluids need to be compatible and the purification of RNA needs to be complete enough as to not inhibit the amplification reaction or confound the amplification reaction with DNA contaminants. Isothermal PCR was selected since the reaction occurs at 70C and often lower, below boiling and reduces chances of evaporation, and makes double stranded DNA less available to be copied. The use of more primers makes this type of PCR more precise and sensitive. Important features that apply to sensing schemes are the formation of magnesium pyrophosphate precipitate causes a visible change in the media and a measureable change in impedance in positive reactions. Additionally, the mRNA needs to get into a well, but not escape when the reaction is started. Timing tools for the initiation of the reaction also needs to be determined.
Current Design:
Ideally we would like there to be multiple wells in parallel to detect many genes at the same time. But, our beginning prototypes will likely have 3 wells: one sample, one positive control and one negative control. It is possible that the current used to sense the reaction, may also keep the reaction components in the reaction cell.
The main challenge of this system for amplification is that the reaction requires special chemistry to work properly and some cell components interfere with PCR. Since all steps happen on one chip, all the fluids need to be compatible and the purification of RNA needs to be complete enough as to not inhibit the amplification reaction or confound the amplification reaction with DNA contaminants. Isothermal PCR was selected since the reaction occurs at 70C and often lower, below boiling and reduces chances of evaporation, and makes double stranded DNA less available to be copied. The use of more primers makes this type of PCR more precise and sensitive. Important features that apply to sensing schemes are the formation of magnesium pyrophosphate precipitate causes a visible change in the media and a measureable change in impedance in positive reactions. Additionally, the mRNA needs to get into a well, but not escape when the reaction is started. Timing tools for the initiation of the reaction also needs to be determined.
Current Design:
Ideally we would like there to be multiple wells in parallel to detect many genes at the same time. But, our beginning prototypes will likely have 3 wells: one sample, one positive control and one negative control. It is possible that the current used to sense the reaction, may also keep the reaction components in the reaction cell.