Overview
Our work in lab this year can be chronologically separated into four main categories — DNA extraction, LAMP, our fluorescent probe assay, and asymmetric PCR.
DNA Extraction
We proposed cellulose dipsticks as a low cost approach to genomic DNA purification. To determine whether dipsticks were capable of transferring sufficient DNA from cell lysate for amplification, we used this approach to purify DNA from E. Coli, then used the extracted sample to amplify the gapA gene via PCR. We grew up E. Coli, lysed them, then dipped the unwaxed end of the dipstick into the solution to make the DNA stick to the paper. After washing the contaminants off with a wash buffer, we ran PCR amplification of the gapA gene, and we tested the success of the dipstick method by running gel electrophoresis to see if the corresponding gapA gene size was visible. We noticed that the dipsticks began to crumble during dipping and washing, so we switched to cellulose squares used with mulitple clean tweasers. This method proved successful, and was thus used in future DNA extraction from HEK293T cells for cellular LAMP.
LAMP
Before moving forward with the amplification from cellular DNA, we had to confirm that we could amplify the F2RL3 fragments via LAMP, serving as our positive and negative controls for further experiments. We designed five primer sets with standard LAMP design software and performed in vitro LAMP on ordered Twist DNA to confirm amplification. Following this success, we opted to perform further confirmatory and optimization testing. This included using SYBR Green to measure dsDNA amplification over time in a qPCR machine, as well as SYBR Safe in gel electrophoresis to confirm the presence of our product (a long band revealing the presence of multiple different-sized concatemer products).
Fluorescent Probes
Now that we could amplify the target gene using LAMP, we proceeded to combine dipstick extraction of F2RL3 from HEK293T cells with the amplification portion of LAMP
Since we confirmed we could amplify the wild-type and mutant F2RL3 fragments in vitro, we began testing our fluorescent probes. We used an online calculator to create a set of probes which bound differential amounts of fluorophore to the SNP region depending on the presence or absence of the SNP. Thus, after amplifying template and cellular DNA, we added a 1 uM mixture of probes and ran a fluorescent binding experiment (as described in our protocols). After initially not seeing promising results, we opted to dilute the amount of DNA mixed with probes and changed the ratios of our probes and sinks.
Asymmetric PCR
We ran a restriction enzyme digest (the Ale1 enzyme) to reconfirm the identity of our product. This experiment, designed to cut the concatemer in the flat region of the DNA, suggested that we needed to switch to a different primer set than the one we had been using for LAMP so far. Repeating probe-based fluorescent experiments did not yield promising results again, which is when we switched to asymmetric PCR, which produces a simple, single-stranded product that might bind to our single-stranded probe and sink sequences more easily.