Get that Genotyping PCR to Work EVERY TIME

Say you just joined a lab and have been assigned your very own project to work on. As part of your new responsibilities, you have to breed and maintain the mutant (or transgenic) mouse line which you will be using for your experiments. An integral part of mouse genetics experiments is determining the genotype of each mouse, which is typically performed through genotyping PCR.

What is Genotyping PCR?

Genotyping PCR is used to determine the genotype of an organism (e.g., WT vs. mutant, or WT vs. transgenic). PCR primers are designed to specifically amplify either a portion of the transgene (in a transgenic animal) or the mutation (in a mutant animal). They are then used in a reaction containing DNA from the animal.

DNA for your genotyping can be extracted from different tissues, depending on what your purpose is. For general colony maintenance, or when using adult animals, you usually remove a small piece of the tail (~2-3 mm) with a pair of scissors. For embryos, depending on the age and experiment, the yolk sac or a limb bud can be utilized for genotyping. However, virtually any small piece of tissue will provide sufficient DNA for your genotyping reaction.

Now, unless your project involves actually generating said mouse line, chances are the lab already has an established genotyping PCR protocol. Alternatively, if the mouse line was purchased from a commercial source, such as Jackson Laboratories, they also provide the primers as well as PCR conditions for genotyping. Therefore, theoretically, genotyping PCRs should work all the time. However, as with everything else in the lab, there is no one technique or experiment that always works. So, here are some tips on how to ensure that your genotyping PCR works as it should, and how to troubleshoot it if it doesn’t.

Prevent Cross-Contamination Between Samples

To ensure that no cross-contamination occurs during sampling, clean scissors and forceps and any other tools with 70% ethanol in between animals. Remember, just a tiny drop of blood or a small snip of hair has enough contaminating DNA to confound your genotyping. When using the yolk sac for genotyping embryos, make sure you wash most of the blood off. Since this is made up mostly of maternal blood, it will contaminate your sample.

In the same note, make sure that you only have one tube open at any given time in between sampling and adding your DNA template to your PCR mix. Lastly, take care of your samples which contain genomic DNA: store them for short-term at room temperature and long-term at 4°C.

Run your CONTROLS

There will never be a good reason or excuse to skip your positive and negative controls. Your positive and negative controls will depend on whether you are genotyping for a mutation or a transgene. If your mouse line contains a deletion/frameshift mutation in a gene, then most likely you will be comparing the difference in size of this DNA fragment between wild-type and the mutant version of the gene. On the other hand, if you have a transgenic mouse line, then your genotyping assay will be detecting the presence or absence of the transgene.

For mouse lines with mutations:

If you are just inheriting the line, positive controls are usually previous samples that have already been genotyped. If you are starting with a new line, then your first few genotyping PCR runs may or may not contain a positive control. If you have a plasmid containing the DNA fragment that you are trying to amplify in the genome, then you can use that as your positive control. However, sometimes this will not be the case, so once you get samples that have been correctly genotyped, store these and use them as your positive controls for subsequent genotyping. Your negative control will be your PCR mix without a DNA template.

For transgenic mouse lines:

You can use a plasmid that contains your transgene as your positive control. However, since your assay will be detecting either the presence (transgenic) or absence (non-transgenic) of a band, you must have TWO negative controls: one without a DNA template, AND one containing genomic DNA from a non-transgenic animal.

For all assays, the negative control is especially important to rule out contamination. Without your negative control, you could be analyzing your samples incorrectly, which will ruin all your downstream experiments! If this happens, you may have to throw away your working primer solutions, change your filter tip boxes, and even change your water.

Follow PCR SOPs

Remember that genotyping PCR, like any other PCR experiment, is extremely sensitive and prone to errors; therefore, it is important to ALWAYS follow PCR standard operating procedures (SOPs). These have been extensively discussed in previous posts in this blog series, but just to summarize:

1. Take care of your primers and reagents.
2. Follow the recommended PCR cycling conditions AND regularly check if your programmed cycle in the machine is still correct!
3. Use the correct % gel for electrophoresis.

If All Else Fails…

Sometimes, it will happen that the reported genotyping protocol just doesn’t work or has never worked in your hands. In that case, you will have to troubleshoot and optimize your genotyping PCR. As with troubleshooting any other PCR experiment, make sure you change only one variable (e.g. primer concentration, template concentration, temperature, etc) at a time.

Good luck, and happy genotyping!