Create a better multiplex genotyping PCR protocol for the identification of Dusp4 WT and KO alleles in mice
I was asked to design a better multiplex PCR protocol for detecting both mouse Dusp4 and KO alleles in the same reaction. Currently available primers give a DNA band size of 647 bp and ~620 bp for the WT and KO alleles respectively. Hence, they are suboptimal for PCR multiplexing. I also found that 2/3 of each amplicon shares the same sequence to each other. This greatly disfavored the amplification of the WT allele in DNA samples from WT/null heterozygous mice probably because of the presence of strong DNA motifs, which may affect PCR amplification efficiency.
Yet another obstacle was that the precise point where homologous recombination took place into the the knockout genome was partially unknown. In order to help, I’ve designed multiple DNA oligonucleotides for PCR amplificaiton of Dusp4 wildtype and knockout alleles.
Within a week, I’ve figured out all of the above, resolved WT allele PCR amplification and succeeded at designing a multiplex PCR protocol for detecting both WT and KO alleles from heterozygous mice, saving time, reagents and labor to the requestant researcher.
As shown in the Genetics and Challenge sections below, there are multiple problems with detecting WT and KO mice by using multiplex PCR.