We had four centrifuges, a thousand Pasteur pipettes, reagent kits, a freezer for minus twenty, a thermal cycler, a phoresis chamber, and a transilluminator that turned on only if you pressed off. The only thing that raised doubts was how we would do PCR in the field, but we knew that sooner or later we would try that too.
We did it for the first time at the Moscow GikPiknik on June 13-14 in Krasnaya Presnya Park. We set up tables, seated the volunteers, welded agarose gel in the microwave from our office (we got beautiful briquettes of jellied meat, transparent as a baby’s tear, for electrophoresis) and started genotyping. I wanted to show everyone that genetics is fun!
The Atlas team was doing genotyping of DRD4, a dopamine receptor gene known as the pleasure hormone. The DRD4 gene is responsible for making our brain cells “sense” the presence of dopamine. The end of this gene is formed by repeating sections. The number of repeats can be different – from 2 to 11. Alleles will be numbered accordingly: 2R, 4R, 7R, etc. Depending on the number of repeats in the gene, the protein will respond more or less to dopamine. This affects the intensity of the feeling of pleasure from events or food and, as a result, the level of excitement of a person. People with seven repetitions (7R polymorphism) need more dopamine to achieve satisfaction, they are unlikely to have enough goodies or similar simple pleasures. Often they have to go on trips for this, engage in risky sports – they do not sit still at all. This is confirmed by studies that have identified a relationship between the 7R allele and the distance of migration of ancient people from Africa to Eurasia. To determine polymorphism, it was necessary to isolate DNA, carry out a polymerase chain reaction (PCR), and drive its results on electrophoresis: arrange races of the DRD4 gene segments in agarose aspic. But first things first. At the stage of DNA extraction from saliva, everyone could take part. First, it was necessary to scrape the inside of the cheek with a cotton swab and rinse it in a test tube with water. Then we rolled the test tube in a centrifuge, and under the action of centrifugal force, the epithelial cells settled and formed a sediment. Pour the water, and add lysis buffer (detergent) to the test tube to destroy the cell walls and heat in a water bath. As a result, instead of whole cells, a smoothie appears in the test tube, like from a blender: the remnants of membranes, cytoplasm with proteins dissolved in it, cellular organelles and DNA. We recover it in three steps. The first stage is based on polarity. All molecules are divided into two groups – polar (that is, having a charge) and non-polar (without any charges on the surface). Polar molecules like polar solvents (this is how salt dissolves in water), and non-polar molecules dissolve only in non-polar liquids (essential oils – in alcohol).
Therefore, we add a non-polar solvent chloroform to the test tube (DNA does not dissolve in it, but cell membranes do) and send it to centrifuge. As a result, we get a two-phase solution: water with a film of DNA on top and chloroform that sank to the bottom. It is necessary to carefully draw out the aqueous part with DNA with a Pasteur pipette and transfer to another test tube, and pour out the chloroform. To the geeks’ credit, we can say that even the youngest guys dared to carry out this jewelry operation. Then we added a sediment solution in order to once again wash the DNA from detergents. DNA is a charged particle, so we added a salt solution with high ionic strength to precipitate it. (At some point, we ran out of it, we had to run to the food court, borrow ordinary table salt and dilute it). As a curtain – shock therapy: add ice-cold 96% ethanol. Ten minutes in the freezer, ten thousand circles in the centrifuge – and we can see the DNA at the bottom of the tube. We drain the liquid, and dissolve the precipitate itself in deionized water. This water does not interfere with the work of enzymes that are used in molecular biology, so it can be used for PCR. The rest took place in our laboratory tent (read – in an open field).
With the help of PCR, we obtained many copies of a section of the very end of our gene. Two primers defined the beginning and end of the DRD4 repeat region. They cut it out and copied it many times using the enzyme polymerase, which reproduces the sequence of nucleotides in the image and likeness of the originally available segment of the gene. This process is called amplification. This all happened automatically in a special amplifier. To determine polymorphism, we conducted electrophoresis – the very races that we have already talked about. Our expert molecular biologist Vera Bashmakova dripped the samples obtained after PCR into the wells of the agarose gel.
Then an electric current was passed through the gel. The charged DNA molecules moved in an electric field, and the speed of their movement depended on the number of repeats in the gene: the more there are, the slower the DNA segment. We added a special solution to the gel, which illuminated the DNA if the gel was illuminated with ultraviolet light on a transilluminator. The distance was measured with a molecular weight marker – and voila, you have the adventurous “slow” allele 7R.
The entire analysis took about two hours (excluding the DNA extraction process).
In just two days of the picnic, about 150 people underwent genotyping, of which 13% were genetic adventurers. Moreover, on average, for the Russian population, the 7R variant is found in only 5% of people . It turns out that the concentration of genetic adventurers at the GeekPicnic is increased. By the way, one of the Saturday adventurers was so carried away by field genetics that he came to us on Sunday as a volunteer. UPD : and in St. Petersburg the concentration of genetic adventurers is lower: only 2.5%. In total, for the second weekend of the Gikpiknik in St. Petersburg, we genotyped 160 people.