Experimental Design

Step 1: Identify the Mutation Region

In this case, the mutation region is the amino acid in the 52nd position of the genome, where the amino acid H changes to a Q. Once the region is identified, we have to flank the region with a forward and reverse primer, and design a probe that can bind to that specific region. If the probe binds, it will light up. Because the mutation identified is unique to a variant of Covid, the probe will only bind if that variant is present. Thus if it lights up, we know what variant it is.

Step 2: Create the Probe and Primer Set

There are two ways of creating the probes and primers. The first is handmade in which we highlight the region of interest and systematically add in locks (+) to raise the melting temperature of the probes and primers. The second way is to generate kmers (subsequences of DNA of size k) through Kevolve. These kmers will be unique to a variant. Thus we can infer that a unique kmer is actually a mutation and we can use that kmer to design a probe.

Step 3: Test the Probe

To test our probe, we set up a simple experiment in which we put in the positive control (variant EG.5.1) and the negative control (Delta). We identified two mutations unique to EG.5.1 and expect the probes to bind to them.

Result:

The result was that it worked! The probe successfully bound to EG.5.1 and didn't bind to Delta. That little uptick at the end for Delta is completely normal, as when PCR is run long enough, any small contaminant could be picked up. What we were focused on was the pattern of separation, seen around cycle 12.

Step 4: Multiplex

To further test our probes, we did something called multiplexing, in which we throw in a bunch of unknown samples and see what is picked up. Once we get the results, we go back and check to see if the variants picked up were indeed the variants the probes were designed to pick up. Thus we found out that the EG.5.1 probe picked up samples 3, 5, and 6, all of which were E.G.5.1 samples!