The methods behind my project

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I am doing a genetics project, and whilst I have come to understand the ‘fundamental’ processes which underlie molecular biology, there are many people out there that don’t. This post thus serves the purpose of teaching those of you who interested, just a little bit about what I do and how I do it. I don’t claim to be an expert nor do I claim to be good at it, but bear with me, and maybe you will come to appreciate just how simple this stuff can be.

My project is using the DNA from spotted skaapsteker (Psammophylax rhombeatus rhombeatus)  tail clippings to determine whether the snakes on the east coast, and the snakes on the west coast of South Africa, are different species. I will admit, Genetics is not exactly the most accessible science, but when you peel away the prestige created by ‘full-of-them-self’ scientists and shed your own self-doubt, you will begin to find the methods can be rather simple, and better yet, fun.

Firstly the tissue has to be acquired and this can be done in one of two ways. Either you sub-sample a specimen in a museum (ie: cut a piece of tissue off a preserved animal) or you capture the animal yourself and cut off a piece of tissue. Due to time constraints, I did not capture my own specimens but rather opted to sub-sample museum specimens from the Bayworld museum in the Eastern Cape and SANBI in the Western Cape. Once you have your sub-samples, you have to log the numbers and further sub-sample your original sub-sample. This is done to ensure that there is back-up tissue, in the event that you lose, or contaminate the sub-sample that you are working with. (FYI: I will use the word ‘sub-sample’ more sparingly from here on out)

Figure 1: To left-Test tubes, containing DNA samples from spotted skaapstekers across the southern Cape. To Right- A skaapsteker tail being sub-sampled.

Following this, the original sample is returned to the freezer, to preserve the integrity of the DNA for future use, and the sub-sample is extracted. Extraction is the process whereby the the DNA is purified and isolated from all other compounds that may be present in the piece of tissue. This is an integral step, because the more pure the DNA, the more likely it will be translated into a usable DNA sequence at the end of the entire process.Firstly the DNA sample is broken up to expose the DNA, then the fats and proteins inherent in the tissue are broken-up, using salt and detergents and finally, the DNA is separated from the solution using alcohol and centrifugation. Basically, The DNA is isolated and purified.

Figure 2: To left- process of adding and removing alcohol in the seperation step of extraction. To Right- Centrifuge used for separating DNA from alcohol

Once extracted, a micro-liter of the DNA solution is removed from the test tube and analysed using a Nano-drop to determine the concentration of the DNA in each test tube. This is done to ensure that the DNA was properly extracted and in addition, enables you to modify the ratio of water and DNA going into the PCR step to optimize the reaction. Once all samples have been nano dropped, the DNA from each test tube is transferred into new tubes and primer, mastermix and water are added to the test tube.

The purpose of a Polymerase Chain Reaction is to amplify the DNA already present in the test tubes and create higher quantities, and longer strands of DNA, that can be analysed in the last step of the process.  The primer which is specific, amplifies a particular gene and the mastermix supplies the building blacks, facilitates the binding process and ensures the right conditions are met for DNA amplification.

In my project, I am analyzing the 16s RNA gene so naturally I used a 16s primer to amplify the gene I was looking for. The entire process takes place inside a PCR machine which cycles the temperatures for specific periods of time in order to facilitate the amplification process inside the test tubes.

Figure 3: To left- Nano-drop, used to test the DNA concentration of samples after extraction step. To right- PCR machine, cycles temperature thereby facilitating the amplification of DNA.

Once the PCR machine has run its course, the test tubes are removed, hopefully containing many copies of the gene you want to sample. To test whether the entire process has worked, the amplified DNA samples from the PCR step are inserted into wells in an electrophoresis gel. When an electric current is run through the gel, the negatively charged DNA molecules are pulled through the gel by the positive charge created on the other end of the gel. The gel is porous so the smaller fragments move further through the gel than the bigger fragments, and thus a ladder of different-sized DNA molecules is created. Once 30 minutes have elapsed, the gel is removed from the gel tank and is placed in a machine that takes a picture of the gel.

If you have have been accurate and everything has worked as it was supposed to, you should get a picture with a row of dark black DNA bands across your gel. A dark band is created if the desired gene has been amplified correctly in a particular sample and a row of bands is created if multiple samples have worked together.

PCR 1

 

Figure 4: Row 1- Ladder of differently sized DNA fragments, against which you can compare your samples. Row 2-6- Bands created by the presence of large quantities of the 16s gene, which bunch together because they share the same DNA fragment size. Row 7- Blank, contains no DNA and is inserted as control to ensure that no contamination is present. This sample had a little contamination as you can see above

The above picture shows the result of 5 successful extractions, with each dark band representing the amplified DNA of a individual spotted skaapsteker. Seen as though this picture was taken a few months ago, the DNA has already been sent to Macrogen, along with 10 other samples, for sequencing.  I am happy to say that from this batch, 9 out of the 15 DNA samples produced viable sequences, and following the successful acquisition of more sequences from more localities, these samples will be used to determine whether there is a need for taxonomic reevaluation in the spotted skaapstekers.

 

picture of sequence

Figure 5: Example of a sequence showing the bases along a  section of an DNA strand in a spotted skaapsteker.

 

 

 

The beginning of my project

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I have loved snakes for as long I can remember. They fascinate and inspire me to read more and more. They very well may be the thing I choose to base my life’s work on one day, and it’s this love that has steered me towards my latest Zoological endeavour: the study of spotted skaapstekers and their distribution In South Africa

Unlike my first project which saw me growing buckets upon buckets of invasive water weeds, this project has me in an air-conditioned lab with finely-tuned scientific instruments – a hop, a skip and a mile away from my previous greenhouse and spade. It may not be as glamorous as birthing a T-rex but it has its perks. Firstly, it doesn’t pay. Secondly, it makes me feel stupid and thirdly, it makes me feel more stupid than the laboratory’s pet fish that eats its own semen. Granted, these are not perks but the semen eating Siamese fighting fish is weirdly frightening and fascinating all at the same time. (P.S. I don’t know if the fish’s diet is completely factual but I have it from reliable sources that it is).

Regardless of the fish and its diet, I believe this project is both challenging and rewarding at the same time because it promises new knowledge and new skills that are tantamount to the arsenal of any aspiring herpetologist.

Thus far I have completed the DNA preparation for most of the DNA samples from the Bayworld museum in Port Elizabeth. The preparation which involves clipping body tissue, extracting, amplifying and running DNA is a rather painstaking and unpredictable process which leaves even the most robust knees shaking from side to side. The end product, a small picture with an array of black and white lines can make or break your day and luckily for me, my day was made the first time I delved into the world of genetics.

My DNA samples were good and for this reason they were packaged and sent to South Korea only weeks ago for further preparation. The product of the preparation carried out in South Korea will be inputted into a rather sophisticated programme to determine whether the samples ascertained from the museum prove or disprove the existence of multiple subspecies of spotted skaapsteker in South Africa.

The project is still in its early stages because I have only done approximately a third of the necessary DNA preparations for the project. The next step which was begun only days ago will involve me preparing spotted skaapsteker DNA samples from SANBI (South African National Biodiversity Institute) in Cape Town. Once prepared, the large batch of samples will be sent to South Korea and following that will be analysed in conjunction with the samples from the Bayworld to determine whether spotted skaapstekers are perhaps more than just one species.

Although I am only at the beginning of this sure-to-be stressful endeavour, I am excited to see what the future holds and whether the spotted skaapstekers warrant taxonomic reevaluation.