In the simulator below, you will see two pieces of 'template DNA'. This represents the CAST gene from both chromosomes of a cow that is heterozygous for the variation we are looking for. The highlighted region of the template DNA is where the PsyI enzyme will try to bind and cut (PsyI recognition sequence: GACN*NNGTC). Using the temperature buttons and the 'create primer buttons', simulate four or more cycles of a PCR reaction.

The simulations above is a shortened version of the actual CAST gene, to make it easier to see.

Below is a version of the same scenario, but this time it has the true, full-size sequence of CAST

In the scenario below, the two DNA sequences represent the PCR amplicons that the CAST primers would amplify from a heterozygous cow. Since the difference between the CAST alleles is not at either primer recognition site, our primers will amplify equally from both alleles. Therefore, subsequently applying the PsyI restriction enzyme will produce a pattern of differentially cut and uncut DNA fragments. Remember that PsyI has the cutsite GACN*NNGTC