The Dopamine Receptor D4 (DRD4) gene contains a segment 48 base pairs long, which repeats between 2 and 11 times, depending on the individual. This pattern in DNA is referred to as a VNTR (Variable Number Tandem Repeat). Studies have shown particular phenotypes, or traits, in people who have exactly 7 repeats of this DRD4 VNTR. Such people may exhibit characteristics such as a novelty seeking personality, susceptibility to ADHD, longevity, a nomadic lifestyle, and other traits.
Michal, Zeb, Noah and Georgia have been developing and troubleshooting a method in the HiveBio lab to determine the number of these repeats in the DRD4 gene of our research participants. They begin by taking a buccal (cheek) swab to gather cells, and then make various dilutions of the cell sample in a chemical buffer that exposes the DNA.
Next they amplify the number of DNA strands in their sample by using a technique called Polymerase Chain Reaction (PCR). The amount of DNA containing the DRD4 gene is so tiny compared to the total amount of DNA in our cell sample, that we need to make more copies of this part of the DNA in order to analyze it. In order to amplify our DNA, we add it to a molecular cocktail of all the ingredients needed to make more copies of the DNA segment of interest. This cocktail includes very specific molecules called “primers” that specify the region of the DNA to be amplified. Design of the primers is one of the first and most important steps for a research study of this nature. Once the DNA of interest is copied over billions of times using a machine called a thermocycler, we have enough to separate it out in a gel.
Some might just see a bunch of tick marks on a clear gelatin-like substance, but to those savvy in molecular biology techniques, this “gel” shows the relative sizes of the DNA being examined. The DNA is stained with a blue dye so we can see how far it has run down the length of the gel. The gel is made in the same way you make Jell-O at home. You heat a powder, in this case agarose, in a certain volume of water, and then allow it to cool in a rectangular mold. In this case, the gel is made from a 2% solution, so 2 grams of agarose powder for every 100mL of water. We put a comb at the top so when the gel solidifies we have wells in which to load our DNA and keep our samples separate.
When solidified, the agarose produces a homogeneous gel matrix through which DNA can diffuse when an electrical gradient is applied. The side of the gel where we load the DNA is the negative end, and the far side is the positive end. DNA has an overall negative charge, and its rate of diffusion through the gel is dependent on the length of the DNA fragment, also referred to as its size. By looking at the relative distance the bands have traveled, we can estimate the size of the DNA fragments we’ve loaded. The smaller the pieces, the farther they will go in a shorter amount of time! Once the DNA had moved most of the way down the gel, as indicated by the blue loading dye, the team stopped the gel electrophoresis and took photos for analysis.
Lanes 1 and 5 are the positive controls, samples of DNA amplified using the control primers. Lane 4 is the negative control, no DNA. Lanes 2 and 3 are DNA samples from participant 1, using primer set 1 and primer set 2 respectively. Lanes 6 and 7 are DNA samples from participant 2, using primer set 1 and primer set 2 respectively. Lane 8 is the DNA ladder, a standard set of DNA lengths.
Ideally, we should be able to determine the number of VNTRs by looking at the length of DNA in our gel as compared to the standard sizes in the ladder. However, not everything always goes according to plan! The wavy lines of the DNA ladder indicate that something is impeding its diffusion through the gel. The team will have to troubleshoot this to get a clearer and more accurate picture.
So how many DRD4 repeats do our two participants have? Stay tuned to find out…
If you are interested in learning more about the DRD4 Project at HiveBio or have questions about developing your own project to work on in the lab, please contact Zeb Haradon or Michal Galdzicki at email@example.com
The Thermocycler used for this project is a GeneAmp9600 generously donated to HiveBio by Rob Carlson.