Proving Genes in Ball Pythons with miniPCR

Ball pythons are a commonly captive kept, and bred, snake that has many naturally occurring “morphs”. The most popular morphs are simple Mendelian inheritance traits, as opposed to polygenic traits, which means they can be identified with technologies that visualize genes. In this article, we describe how anyone has access to the technologies that make this possible with a focus on do-it-yourself over commercialized testing. Skip to the section on available tests if you would prefer the convenience of those options.

Fast Genetics Primer

The focus of most of article will be on Mendelian Inheritance. This is a model of how genes are passed to offspring based on work from Gregor Mendel. In this model we expect to see 50% of the offspring inherit a specific gene from their parents. This is the same thing that is modeled by Punnett Square, World of Ball Pythons’ Genetic Wizard, MorphMarket’s Gene Calculator, and more. You can even do this calculation right on your fingers as demonstrated in this video.

The sex of offspring is another area where genetic testing can be very helpful and isn’t all that complex. For example, someone may conduct an experiment looking for a sequence normally present in the Y chromosome of an animal. If present, then that animal is male. This also follows the patterns described above — males have X and Y (XY) chromosomes while females have only X chromosomes (XX). Both parents have an equal 50% chance to pass down their specific chromosome and therefore there is a 50% probability they will inherit the Y chromosome and be male. Being able to identify male and female animals through genotyping is useful for a variety of reasons: identifying animals that do not have a strong sexual dimorphism (you can’t see the difference) or animals that can’t be otherwise sexed (via inspection, example: venomous animals). Genotyping animal sex will not be covered in this article.

The term polygenic was already used above. Polygenic Traits are traits that are influenced by interactions between several genes. These traits would be the ones that may modify eye colors or create aberrant patterns on the animal (or human, of course) but are difficult to recreate when breeding because several genes must be inherited. This article will not include much discussion on polygenic traits, however it is important to contrast the difference. Traits that follow Mendelian Inheritance are easy to predict while Polygenic Traits are not — this would be where breeders would create “lines” of animals (such as specific dog breeds, as an example).

PCR (polymerase chain reaction), in a nutshell, is a way to amplify specific portions of DNA to make it easier to identify. PCR is a very old technology that emerged in the 80s. Since then, it has been the forefront in early detection of causes of disease whether the cause is inherited or introduced by a pathogen such as a virus or bacteria. Since PCR is such an old technology that is a staple in biology it is very easy to find a wealth of information about PCR. The pervasiveness of this technology, the cost efficiency of it, and the accessibility we have to it makes it a very good candidate for classroom and home exploration of biology topics.

miniPCR

miniPCR is an amazing company that offers easier access to biology topics. You can shop through the equipment they offer as well as complete classroom kits. miniPCR has even found a place on the International Space Station. Even working with reptiles isn’t something new for miniPCR — they’ve done that, too! The miniPCR team is great to work with and all of them are experienced in the industry.

The equipment used in my experiment at home all came from MiniPCR with their help.

Special thanks to Allison Nishitani and Sebastian Kraves from miniPCR for helping me to get my experiment to work with miniPCR.

miniPCR Order List

Below is a list of the equipment I purchased from miniPCR. Unfortunately, if you’re viewing this on the Medium website, there is not a great way to include a table and the formatting will be a bit odd. These are not affiliate links or anything similar — these are direct SKUs available on the website with no association to the author.

If you directly contact miniPCR they will most likely prepare an order for you if you ask rather than having to click through this list. Sending them the image directly may also be helpful just in case I’ve left something off or included something unnecessary.

With some luck, enough of us will be interested in this process that they may even make a kit for people genotyping snakes!

Tests Being Done Now

If you would like to skip the investment of time and money in performing experiment that will be described here then you do have options.

You can use one of the services below to identify genes rather than doing it yourself. This should be much more accessible to most keepers.

Ball Python Genetics Project

Dr. Hannah Siedel operates the Ball Python Genetics Project with her students at Eastern Michigan University. Using available research, a lot of “elbow grease” in repeating PCR labs, and through contributions across the reptile breeding industry, they have been able to isolate genes and recreate experiments to identify those genes through technologies like PCR.

You may even recognize Dr. Hannah Siedel from podcasts like Samson Rivera.

All of the research and emerging identifications from the Ball Python Genetics Project are “open” — that is, it is public research you can go look up yourself and recreate at home (if so inclined). There is no retail test available here. Donations of sheds and identifications are used to further research for everyone.

Rare Genetics, Inc.

Rare Genetics, Inc. has been a staple in the industry for offering genetic testing involving animal sex. This has been a useful service for venomous keepers and keepers who prefer not to “pop” or “probe” animals to determine their sex.

Recently, Rare Genetics, Inc. has announced they will soon have a retail test for ball python genetics.

Rare Genetics, Inc. will have a very useful retail service for identifying animal traits. These will be commercialized tests and, possibly, will not be available in the public space like the Ball Python Genetics Project.

Experiment Notes: Lavender Albino

This test has been successfully reproduced by the author of this article.

Notes Regarding Experiment Changes

You will need to sort out how you plan to extract DNA from the animal you want to test. There are many resources, especially at the Ball Python Genetics Project, that detail how they extract DNA from sheds.

For my experiment, we took a tiny shortcut and used a cheek scrape to extract DNA. This is much easier and much quicker to process.

You will need appropriate primers, ladders, and protocols for your specific test. The product of the PCR experiment may be larger or smaller than the products in the Lavender Albino experiment — which means you need to match them appropriately. That also means that the miniPCR protocol may need different temperatures, lengthier stages, etc.

I would be glad to continue to produce articles on how to perform these tests at home as possible, however, it is time and cost prohibitive in a hobby that very much resists change. :)

Cheek Scrape Method

It is very important to understand that you are not trying to draw blood.

Using a toothpick, rub the toothpick inside of the snake’s mouth for a few seconds. The goal is that the rough toothpick will collect any loose skin cells and biological material we can use for this test. The side of the toothpick can be used to avoid scratching the animal with the tip of the toothpick. This should be minimally invasive for the animal.

The toothpick can then be dropped directly into the miniPCR DPX Buffer for 10 seconds. Discard the toothpick and incubate at 95C for 10 minutes with the miniPCR in Heat Block Mode.

Primer Preparation

This is a step that is, again, a little dependent on what experiment you are doing. When acquiring primers I received primers that were 100uM but need to be used at 200nM. This required dilution. This step will most likely be required but may have different dilution ratios depending on your experiment.

PCR Program

Different experiments will require different PCR programs.

The miniPCR device is controlled by your smart phone. You are able to create experiments and save those experiments.

As you can see in the screenshot above, I have a few saved protocols including a simple Heat Block mode (explained above).

Save your work!

The Complete Experiment

This should be an overview of the whole experiment. Please understand that it is possible I have left steps out or modified them for myself. If you find an omission please let me know ASAP.

All credit for even being able to complete this experiment goes to miniPCR, Allison Nishanti (miniPCR), Sebastian Kraves (miniPCR), Dr. Hannah Siedel (Ball Python Genetics Project, Eastern Michigan University), and all of the researchers that made this possible.

Primers and Experiment

This is based on A community-science approach identifies genetic variants associated with three color morphs in ball pythons (Python regius) (link)

Primer design
217F (5’-GGA GAG AGA ATC CAA CCC TTG -3’)
166R (5’-TGG GTG GCA AAC AAT CAT AA-3’)
188R (5’-CAA AGA CCA TTG TCC ATT TCC-3’)

When sourced from IDT (Integrated DNA Technolgies) I had to dilute the primer mix with 470ul nuclease free water (1:50 dilution) to reduce them to 2uM primer. Since this experiment requires 3 primers, this is required for all 3 primers.

This diluted primer mix will now be called “Primer Mix” for the rest of this article.

Agarose Gel Preparation

You will want to do this part sooner than later so that the gels have time to cool and cure. You can keep gels in an airtight container at room temperature for later use. You can also cut gels if you only have a few samples to run at a time. Prepare the gels early.

I used the SeeGreen product from miniPCR.

The video linked above should explain the steps required including how to mix and cast the gels, why they are used, and what the expected outcome is.

Be very careful loading your gel.

Collecting DNA

As discussed, you can source the materials required and reduce shed to DNA samples. For convenience and time, I was able to source DNA through swabbing the snake’s cheek gently with a toothpick.

DNA Preparation
Gently swab snake with toothpick
Drop the toothpick in a PCR tub that contains 50ul miniPCR DPX Buffer
Wait 10 seconds, swirl lightly
Discord toothpick
Close PCR tube, load into miniPCR thermal cycler
Set to heat block mode at 95C for 10 minutes
Run cycle, let cool at room temperature while preparing mix

This product will now be DNA Sample for the rest of this article.

Pipette, Cry, Repeat

As a forewarning, this process can be very cumbersome. You will need to measure exact amounts of liquids using the micropipettes. That may include a lot of dial spinning. You will want to change tips between each sample and never contaminate your mixes with other agents or DNA.

To save time you will want to create a single batch mix of everything that each sample needs. This will be a large tube full of 8x the amount of agents you need (without the DNA). For example, if each sample includes 3 ul of primer mix, I add 24 ul of primer mix to a tube along with 48 ul of 5X Master Mix, etc, etc. Once mixed, you can then use a micropipette to divide and dispense that liquid into 8 smaller PCR tubes.

For best results, try to get the micropipette tip in the PCR tube close to the wall of the PCR tube and not far from any liquid already in the PCR tube. The dispensed liquid should all fall into the tub and be collected at the bottom.

During the last step of dispensing all liquids you may want to take a moment to “suck up” and “spit out” (that is, move the plunger up and down on the micropipettte a few times) the liquid in the PCR tube to mix it together. You can do this both during Master Mix creation as well as when you add the DNA sample.

Creating Reaction Mix

The reaction mix is detailed below. Note that “water” should be nuclease free water. For this step it is very convenient to have multiple micropipettes already dialed to the appropriate amounts (but that can quickly get expensive).

You should make a control containing no DNA and only water to make sure you don’t have any contamination, that you have loaded the gel correctly, etc.

Unfortunately, if you are reading this on Medium, there is no good formatting for tables. You probably want to cut and paste this into something else.

Sample
13 ul water
6 ul 5X Master Mix
3 ul Primer Mix
8 ul DNA

30 ul total volume

Control
21 ul water
6 ul 5X Master Mix
3 ul Primer Mix
0 ul DNA

30 ul total volume

The ratio of water to DNA, etc, may be tweaked for your individual experiment. This worked well for me. Alternatively, you may try 4 ul DNA and 17 ul water, for example.

Make sure you did not skip the section above regarding a bulk mix. This will save you time and can avoid many errors.

miniPCR Program

PCR is a little bit of an art and you may want to tweak some of these values depending on your specific experiment, the primers used, etc.

Here is a screenshot of the program that worked best for me.

Initial Denaturation: 94C, 120 seconds
Denaturation: 94C, 30 seconds
Annealing: 57C, 30 seconds
Extension: 72C, 60 seconds
Number of Cycles: 35
Final extension: 72C, 300 seconds

Be very intentional when entering these values. Any mistakes will waste a very large amount of time.

As mentioned, you may tweak this. You can try fewer cycles if the visualization looks good. If not, then you may try more cycles. This is an exercise left up to individuals with more experience with the PCR process.

Loading Samples for Visualization

I used 10ul of ladder in the first lane, then 15ul of PCR product in all remaining lanes with the final lane being the control product (the one with no DNA and only water).

I loaded this into the blueGel electrophoresis system by miniPCR.

Be very careful when loading liquids. It might be worthwhile to explore YouTube videos on how to do this. You do not want to load too shallow (the liquid ends up on top of the gel) or too deep (the liquid ends up underneath the gel). It needs to be loaded directly into the well.

Once loaded, you will run the blueGel Electrophoresis system for at least 20 minutes. After 20 minutes you should start seeing the ladder lane separate. The other lanes may not appear to separate yet, but this step requires patience.

Results

The “wildtype” reaction should be a 429bp product.
The “het lavender albino” reaction should be a 349bp product.

Animals with no lavender albino gene should have a single line.
Heterozygous Lavender Albino animals should have two lines.
Lavender Albino animals will have only one line.
The control lane should end up with a line all the way at the bottom.
Other lanes will also have product pooled at the bottom like the control lane — ignore those.

Use your cell phone and the included shroud to photograph your results!

Conclusion

Is gene testing the future of ball python breeding?
Actually, it’s the present!

Breeders are already using this technology to plan breeding seasons. This method will reduce the time it takes to “prove out” a snake since you can genotype that animal and focus on animals that have specific traits you want. For ball pythons, which can take 3 years for a female to reach sexual maturity, you can optimize your programs in hours instead of years.

In the future, it should be entirely possible to multiplex some of these tests together and “bulk test” animals for many different traits.

As mentioned above, you may also seek commercial testing from a third party like Rare Genetics, Inc.

Did you get results? Let me know!