JAX Genotyping Protocols Explained: Standard PCR and Melt Curve Analysis

Your JAX mouse strain has arrived in your facility, and you are ready to start genotyping.  What primers and reagents do you need to purchase?  What are some of the results that you can expect from genotyping?  What are the optimal conditions to successfully run this protocol?

Let’s walk through JAX genotyping protocols for standard PCR and melt curve 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.

Overview of JAX Protocols

Here is a list of the information that you can expect to find on a JAX Genotyping Protocol.

Section

Description

Expected Results

A description and example of the results you can expect when running this protocol

Protocol Primers

A list of the primers needed for this protocol, including the full sequence

Reaction Components

List of the reaction components (i.e. polymerase, dNTPs, buffer) that are used for this protocol

Cycling Parameters

A description of the PCR cycling conditions that we use at JAX

Strains Using This Protocol

A list of the other strains using this protocol, used to determine specificity

 

Section Details

Let’s take a look at a standard PCR protocol for an Interleukin 10 (Il10) knockout as an example of what kind of information you can find on a JAX genotyping protocol. 

At the top of the protocol is the strain name, strain stock number, protocol ID, protocol name, and version. Below, the Notes section provides 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 on an agarose gel, with the primers and cycling parameters that are listed.

Detailed Description: A standard PCR protocol will show the expected band sizes and show an example of a gel that was run at JAX. Please note that the genotypes that you see on the example gel may not be the same genotypes of the mice that are distributed from JAX.  For example, even if we only distribute homozygous animals from JAX, you may see wild type or heterozygous samples on the example gel.

How you can 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.

Expected Results FAQ:

Q: What does “PHet” mean?

A: “PHet” refers to a pseudo-heterozygous sample. This control may be included when only homozygous and wild type DNA is available, and is produced by mixing the DNA from those genotypes together. This is often seen in mouse strains where only the homozygous genotype is available for distribution.

Q: Why can’t this standard PCR protocol differentiate hemizygous and homozygous transgenic animals? How can I differentiate them?

A: Standard PCR and melt curve protocols for transgenic strains are often only able to determine the presence or absence of the transgene. This is because transgenes integrate randomly into the genome, and often it is unknown where the transgene integrated. It is impossible to design a wild type primer set without that sequence information, therefore you cannot differentiate hemizygous and homozygous animals with standard PCR. For such transgenic strains, you must use a QPCR protocol to differentiate them.

Protocol Primers

Overview: Perhaps the most important information on the JAX protocol is the sequence of the oligonucleotide primers. The section lists the protocol primers, with their primer ID, sequence, and description of the primer type.  

Detailed Description: JAX genotyping protocols will have a range of primers listed – there will be a minimum of 2 primers, but most protocols will have 3-4 primers listed. You will see that each primer has a primer ID and the primer sequence. In this protocol, you see 3 primers labeled “Mutant Reverse”, “Common” and “Wild type Reverse”.

The Primer Type is a description of whether the primer is “wild type” or “mutant”, and often whether it is a reverse or forward primer. Some primers will be labeled as “common”, which means that it works in combination with both the wild type and mutant primers. The Reaction (A or B) indicates what reaction each primer should be included in. If all of the primers are run in reaction A, then it means the reaction is “multiplexed”. If the primers are split into reactions A and B, it means the protocols should be run “separated”.

You may also be able to find more information in this section on where the primers anneal. For example, in this protocol for a Syn1-Cre strain, under “Note” we can see that the transgene forward primer anneals to hSyn and the transgene reverse primer anneals to Cre. There is also an internal positive control (IPC) primer set that binds to a gene that is expected to have a band in every mouse DNA sample. We use several different IPC primers at JAX, and select them based upon their size so that they are an appropriate control for the corresponding mutant primer set.

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. This section may also provide useful information on where the primers anneal, which will help determine how specific the genotyping protocol is.

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 standard PCR and melt curve protocols, typically all primers need to be used to amplify both the wild type and mutant products.

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 band. Typically, IPC primers are used when it is not possible to have a wild type primer set, for example for a transgenic strain. If the genotyping protocol includes IPC primers, we recommend that you use them because it demonstrates that you PCR is working and can help with troubleshooting if any problems arise.

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

 

Overview: 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 (left) and cycling parameters (right) that are used at JAX for standard PCR protocols.

Detailed Description: This section includes all of the reagents, including the Taq polymerase, free nucleotides (dNTPs), buffer, and more. 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 PCR reaction. In this section, you’ll find the cycling parameters that we use at JAX. This includes the cycling steps for the annealing, extension, and elongation steps. We have included the temperatures and cycle numbers for each of these steps.

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 PCR in your facility. There are many PCR setups that will be useful, so if you’re already doing PCR feel free to use your current setup with JAX primers. 

Reaction components and cycling parameters FAQ:

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.

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: What polymerase does JAX use?

A: Kapa 2G Fast Hot Start (HS), Catalog # KK5519. This polymerase is used for all Standard PCR protocols and Melt Curve analysis protocols.

Q: What is the glycerol used for in the reaction?

A: Glycerol is used as an additive to prevent evaporation and make the amplified product more stable to prevent the formation of secondary structures. More information on glycerol and other PCR additives can be found here.

Q: What final reaction volume should I use?

A:  Your final reaction volume will depend on the size of the PCR tubes that you are using. Reaction volumes are typically in the range of 20 – 50 μL.

Q: How do I extract/isolate DNA from mice?

A: JAX provides several protocols for DNA isolation, however if you already have a protocol that you’ve used for other tissue samples feel free to use that here.

Q: What is a “touchdown” protocol?

A: You may have noticed that we use what is called a “Touchdown” protocol for all of our strains. This means that instead of choosing a single annealing temperature, we start at a high temperature (65 degrees) and decrease the temperature every cycle until you get to 60 degrees. Since we run many samples in our facility, this touchdown protocol allows us to use the same cycling conditions for every strain. If you do not want to use the touchdown protocol, or your thermocycler does not have a program for it, then you can choose to use a single annealing temperature. You may need to test multiple annealing temperatures to find the one that works well for that particular primer set.

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 Il10 knockout, you can see that all 10 of the mouse strains listed express the same Il10tm1Cgn allele, suggesting that this protocol is specific for that allele.

 

 

Additional Considerations for Melt Curve Analysis Protocols

The melt curve analysis is a protocol that is based on a standard PCR assay and incorporates fluorescent dye and a melting protocol following traditional PCR cycling. Distinguishing the different PCR products is based on the relative fluorescence intensity with amplicon melting. We use melt-curve analysis because it can save time and money when performing large-scale amounts of genotyping. While very similar to standard PCR protocols, there are a few additional considerations and method differences with melt curve analysis. Need an introduction to melt curve analysis before moving forward? Review this introduction to advanced PCR methods for more background information.

 

While the primers section for a melt curve analysis will be the same as for a standard PCR, the results section will tell you what results to expect when you run this protocol as a melt curve analysis. This information is provided in the form of melting temperatures, or Tm. Each peak corresponds to an amplified product with a characteristic melting temperature. In this protocol, the samples in red have a single peak at 83°C, which corresponds to the internal positive control (IPC) product. The samples in green have two peaks, one at 83°C for the IPC and one at 86.5°C that corresponds to the Cre transgene.

To run a melt curve analysis protocol, you will need to perform the PCR reaction in the presence of an intercalating dye, and use a thermocycler that can measure melting temperature.

Melt Curve Analysis FAQ:

Q: What intercalating dye does JAX use for melt curve analysis protocols?

A: We use EvaGreen Dye but you can also use SYBR Green, or other double-strand specific dyes. This paper discussed other options: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095797/

Q: What thermocycler can we use for a melt curve analysis?

A: We use a Roche Light Cycler 480, however any thermocycler that can measure melting temperature should be sufficient.

 

Q: I am seeing different melt curve temperatures (Tm) when I run this protocol in my facility. What is going wrong?

A: Melting temperatures can be variable, and may not be the same when run in a different facility with a different PCR setup.  We provide the melting temperatures as a starting point, however best practice is to run samples with a known genotype (wild type, heterozygous, homozygous) and use those to determine the melting temperature with your PCR setup. Do not be surprised if your melting temperatures are several degrees different from what we have seen at JAX!

Q: Do I have to run this protocol as a melt curve analysis? Or can I run it on a gel instead?

A: Even if a protocol is described to be a melt curve analysis, you may be able to run it on a gel instead. However, some primers sets work better as a melt curve analysis, and that may be noted on the protocol if so. If you do attempt to run the primers out on a gel instead, we recommend running the protocol separated for the first time to ensure that you’ll be able to differentiate the products on a gel.