Biology Asked on February 15, 2021
In this imaginary experiment, I am using T-DNA to make an insertional mutagenesis that will get randomly inserted into the DNA where it might get inserted within a gene, disrupting it.
In order to get homozygous individuals, If I working on plants, I could self-pollinate this particular plant that has had this gene disrupted making homozygous offspring.
In order to prove that, I can do a PCR using 3 primers.
My question is, why 3 primers? I would use 4 primers!
I have been researching it and trying to understand it for myself for a while, but I do not reach any positive conclusion. I think it should be pretty basic and I am missing something.
I think I need forward and reverse primers for the T-DNA, but what about the 3rd primer? If I only use these two primers, though, I will not be able to distinguish between the homozygous and heterozygous.
Thanks!
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