JAX Genotyping Protocols Explained: qPCR, Probe, and Endpoint Analysis

Your JAX mouse strain has arrived in your facility, and you are ready to start genotyping. You realize that a standard PCR won't be sufficient for your application, so you're looking into either a qPCR, Probe, or Endpoint genotyping protocol. What primers and probes do you need to purchase? What are the optimal conditions to successfully run this protocol? There are no agarose gels here - so how do you interpret the results?

Let's walk through JAX genotyping protocols for qPCR and Probe/Endpoint analysis so you can easily find information that will help you genotype your mouse strain. We'll also address some frequently asked questions on JAX genotyping protocols to get you started. Since qPCR and Probe/Endpoint analysis use similar techniques, we'll address these protocols together in this article. Do you need an introduction to qPCR and Probe/Endpoint PCR? Review this introduction to advanced PCR methods for more background information.

Overview of JAX Protocols

Let's walk through a JAX genotyping protocol so you will know how to find information that will help you genotype your mouse strain. Here is a list of the information that you can expect to find on a JAX qPCR or Probe/Endpoint genotyping protocol.

Section Details for qPCR

You have determined that you need a JAX qPCR genotyping protocol so that you can distinguish between hemizygotes and homozygotes for a transgene. Let's take a look at a qPCR protocol for the Tcra transgene in the OT-I transgenic mice as an example of what kind of information you can find on a JAX qPCR genotyping protocol. At the top of the protocol is the strain name, protocol ID and name, and version. Below that, the notes provide pertinent information from The Jackson Laboratory genotyping group about this protocol.

Expected Results

Overview: The Expected Results section will tell you what results to expect when you run this protocol with the primers and cycling parameters that are listed. For qPCR protocols, you will typically see cycle threshold (CT) values, which is the cycle number at which the fluorescence is detectable at higher than background levels.

Detailed Description: Quantitative PCR reactions distinguish hemizygous from homozygous transgenic mice by measuring gene dosage. Regardless of transgene copy number, homozygous transgenic mice carry twice the number of hemizygous mice. Zygosity can be determined by normalizing the amount of a transgene-specific PCR product to that amplified from an endogenous mouse gene, otherwise known as the "delta CT" or ΔCT.

For this protocol, you'll see the average ΔCT values. The no-template controls (NTC) have a ΔCT of 0.0 because they are used as the reference value, whereas homozygotes have a ΔCT of 1.5-2.0, and hemizygotes have a ΔCT of 2.7-3.2.

How can you use the information in this section? This section will inform you on the expected results, however please keep in mind that the expected results are based on what we have seen using this protocol at JAX, and may vary with different PCR reagents and thermocyclers. Therefore, you will need to run control samples with known genotypes to determine the cycle threshold with your PCR setup.

Expected Results FAQ:

Q: Why are there no examples of running the primers out on an agarose gel?
A: qPCR protocols are not run on an agarose gel. If the primers were run on an agarose gel, you would not be able to differentiate hemizygous and homozygous genotypes. A real-time PCR instrument must be used to determine the CT values to determine the genotypes of the samples.

Q: How do I interpret the cycle threshold values? How does that tell me the genotype?
A: In qPCR, the amount of fluorescence from both the transgene and reference products is measured at each cycle until they can be detected above background. This cycle is called the cycle threshold (CT). To normalize the amount of transgene product in a given sample, the CT value for the reference product is subtracted from that of the transgene product, producing the "deltaCT" (ΔCT; i.e. ΔCT = CT_Tg - CT_ref).

Because homozygous transgenic mice have 2X the transgene dosage as a hemizygote, their CT should be one cycle earlier, and their normalized ΔCT value should be approximately -1.0 relative to a hemizygote (see example results). Incidentally, because non-carriers from a transgenic colony don't carry any transgenic product to amplify, their ΔCT values are very large negative numbers (not shown).

Q: Why are there no results listed?
A: A qPCR protocol may have minimal, or absent, expected results section. This is because the cycle threshold (CT) values will be variable, therefore what we see at JAX will likely not be useful information for the interpretation of your qPCR results. You will need to run known controls every time you run the protocol in your facility. You should include, at a minimum, a no-template (water) control and a control for every genotype that you need to differentiate (in this example, hemizygotes and homozygotes).

Primers

Overview: Perhaps the most important information on the JAX qPCR protocol is the sequence of the oligonucleotide primers and the Taqman probes. JAX qCPR genotyping protocols will have a range of primers listed – there will be 2-4 primers listed, and all JAX qPCR protocols will have two probes listed.

Detailed Description: The following section lists the protocol primers and Taqman probes, with their primer ID, sequence, and description of the primer type - in this case Transgene Forward, Transgene Reverse, Tg Probe, Internal Positive Control Forward, Internal Positive Control Reverse, and IC Probe. There may be additional information included, such as the reaction the primer should go in (A or B) or any additional notes that may be relevant.

The Taqman probes carry fluorescent tags at their 5' end a "quencher" at their 3' ends so there will be no fluorescence unless the 5' tag and 3' quencher are physical separated. If the transgene target is present, the transgene-specific probe will bind, and during amplification the fluorescent tag will be separated from the quencher, resulting in a fluorescent signal. Each probe carries a different fluorescent tag so the transgene and the IPC products can be differentially quantified.

How can you use the information in this section? You can provide the primer sequences listed on the protocol to a vendor that supplies oligonucleotide primers and Taqman probes. The specific fluorescent tags and quenchers that JAX uses are not listed because you will need to use fluorophore/quencher combinations that will work with your equipment.

Primers FAQ:

Q: Does the Jackson Laboratory sell primers?
A: No, The Jackson Laboratory does not sell primers. You will need to provide the sequence of the primers to a company that does sell primers. Please note the primer ID's are generated for JAX internal tracking and cannot be used for ordering from us or a third party vendor.

Q: Do I need to use all the primers listed?
A: Yes! For qPCR protocols, it is necessary to use all the probes and primers listed to successfully run the analysis.

Q: What are IPC primers? Do I need to use them?
A: IPC refers to Internal Positive Control. IPC primers are designed to amplify a region of DNA on a mouse housekeeping gene, so that all mouse DNA samples will have an IPC product. For qPCR protocols, the IPC must be run to have accurate quantification of the genotype.

Q: What fluorophore and quenchers should I use for the probes?
A: You will need to start by determining which fluorophore/quencher combinations will work with your equipment. At JAX, we use Black Hole Quencher 1 for the quencher for both probes. For the fluorophore, we will typically use FAM for the mutant/transgene probe, and either HEX or JOE for the IPC or wild type probes.

Q: Is this primer set generic, or specific for this strain?
A: There are several places that you can find this information. If the primer set is generic, it will likely be indicated in the protocol name or in the primer notes. For example, a protocol that amplifies GFP will be named "Generic GFP" and will amplify a band for any strain that expresses GFP. This can also be determined in the "Strains Using This Protocol" section. If there are many strains listed, likely it is a generic primer set.

Q: How can I determine where the primers are binding?
A: The Basic Local Alignment Search Tool (BLAST) database can be used to identify where primers bind in the genome. Review of the primary reference from the donating investigator who developed the mouse strain may also provide information on primer binding sites. See the "References" tab on the strain data sheets for some key references.

Reactions Components and Cycling Parameters

In addition to the primers, you'll also need to know what reaction components you will need for your PCR reaction, and what cycling parameters to use. This section includes the reaction components and cycling parameters that are used at JAX.

Description: This section include all of the reagents, including the Taq polymerase Kit, primers, and probes. You will also find the final concentration of all of the reaction components. We have not included volumes because that will depend on your desired final volume and the starting concentration of your reagents. Some protocols will have all primers together in a single reaction "A", and some protocols may have primers split into two reactions "A" and "B".

Once you have located the primers and reaction components, you'll need to know how to run the qPCR reaction. In this section, you'll find the cycling parameters that we use at JAX. We have included the temperatures and cycle numbers for each of these steps. The cycling times are not provided, as you should use the times recommended by your Taq polymerase.

How can you use the information in this section? This section should be considered as a starting point that can be helpful if you're not already doing qPCR in your facility. There are many qPCR setups that will be useful, so if you're already doing qPCR feel free to use your current setup with JAX primers.

Reaction components and cycling parameters FAQ:

Q: What thermocycler can I use for qPCR?
A: We use a Roche Light Cycler 480, however any thermocycler that can measure melting temperature should be sufficient.

Q: Do I need to use the recommended reaction components and cycling parameters?
A: If you already have a PCR setup in your facility that works well for you, we recommend continuing to use the recommended reaction components for your setup and simply use the primers from the JAX protocol.

Q: Why are the cycling times not listed?
A: Cycling times will vary depending on which Taq polymerase you use. For this information, you should refer to the product literature for your Taq polymerase. The polymerase that we use at JAX is very fast, therefore, the cycling times that we use are typically much shorter than with other setups.

Q: Why do all JAX protocols use the same reaction components and cycling conditions?
A: Since we run a high-throughput facility, we have developed a protocol utilizing reagents and cycling parameters that work for all JAX strains. For this reason, it is not unexpected that you may need to optimize portions of the protocol like the extension time or annealing temperature to get it to work well in your facility.

Strains Using This Protocol

Finally, at the bottom of the genotyping protocol you will find a list of all of the strains that utilize this protocol with links back to each strain's corresponding strain datasheet. This section can help you determine what other strains this protocol can be used for, and will give you some sense of how specific this protocol is. For example, if the protocol is for a generic Cre strain, you will see many Cre strains listed in this section. For this Tg(Tcra) qPCR genotyping protocol, you can see that this is the only strain that uses this protocol, suggesting that this one is specific.

Section Details for Probe/Endpoint Analysis Protocols

JAX Probe assays are created by converting existing standard PCR assays to using Taqman probes, which eliminates the need for running agarose gels. JAX Endpoint assays use Taqman probes to genotype for mutations (base pair changes, small insertions or deletions) that cannot be distinguished using Standard PCR. JAX Probe and Endpoint assays use the same equipment, reagents, and data analysis.

Probe and Endpoint assays will use the same equipment and reagents as a qPCR assay. In fact, these assays will look almost identical to the JAX qPCR assays in the primers, reaction components, and cycling parameters section. However, the data for Probe and Endpoint assays is analyzed differently; therefore, there will be different information in the expected results section.

The Expected Results section will tell you what results to expect when you run this protocol with the primers and cycling parameters that are listed. Unlike qPCR assays, instead of monitoring fluorescence levels at every cycle, for Probe and Endpoint assays fluorescence simply is measured at the end of the PCR cycles. For each sample, the fluorescence intensity of the wild-type-specific fluorescent tag is plotted against that of the mutant tag. Therefore, a Probe or Endpoint analysis protocol will show a Fluorescence scatter plot. While the fluorescence values will vary, you can always expect to see that homozygous mutants and wild-type mice are clustered along their respective axes, whereas heterozygotes cluster along the diagonal.