Question

If you had to analyze bands on an agarose gel, can you differentiate a homozygous knockout, homozygous wild type and heterozygous genotype if you are given the different sized PCR fragments for knockout and wild type fragments?

Answer 1

To solve this we have to keep in mind that to determine gene knockout, we have to identify a section of our gene representing the wild type and the same should be absent in the knockout condition in the PCR product and this PCR product should be generated by the same pair of primers as that of others. Another way to find out the absence in KO condition by looking for a shorter PCR product in the KO compared to wildtype.

For wildtype homozygous condition we will get a single PCR product i.e., we will see one strong band of the size of our wild type PCR product.

For KO homozygous condition, we will get one strong band generally smaller than wild type (in the gel it appears to be present below homozygous wild type).

For heterozygous condition, we will get two bands: one at the wild type size and one at the KO size.