Genotyping geeks: field genetics experience

    We had four centrifuges, a thousand Pasteur pipettes, reagent kits, a minus twenty freezer, a thermal cycler, a foresake chamber and a transilluminator that turned on only if you press off. The only thing that raised doubts was how we will do PCR in the field, but we knew that sooner or later we would try this.

    We did this for the first time at the Moscow GikPiknik on June 13-14 in the park Krasnaya Presnya. We set up tables, planted volunteers, welded agarose gel in the microwave from our office (we got beautiful briquettes of electrophoresis, transparent as a baby’s tear) and started genotyping. I wanted to show everyone that genetics is fun! Atlas



    Teamdid genotyping of DRD4, the dopamine receptor gene known as the pleasure hormone. The DRD4 gene is responsible for making our brain cells “feel” the presence of dopamine. The end of this gene is formed by repeating sites. The number of repeats can be different - from 2 to 11. Alleles: 2R, 4R, 7R, etc. will be numbered accordingly. Depending on the number of repeats in the gene, the protein will respond to dopamine more or less. This affects the intensity of the feeling of pleasure from events or food and, as a consequence, the level of excitement of a person. People with sevenfold repetition (7R polymorphism) need more dopamine to achieve satisfaction, they are unlikely to have enough sweets or similar simple pleasures. Often they have to travel for this, engage in risky sports - they do not sit still. This is confirmed bystudies that have identified a relationship between the 7R allele and the distance of migration of ancient people from Africa to Eurasia.

    To determine the polymorphism, it was necessary to isolate DNA, carry out a polymerase chain reaction (PCR), and run its results on electrophoresis: arrange the DRD4 gene segments in agarose jelly. But first things first.
    At the stage of DNA extraction from saliva, everyone could participate. First, it was necessary to make a scraping on the inside of the cheek with a cotton swab and rinse it in a test tube with water.
    Then we rolled the tube in a centrifuge, and under the action of centrifugal force, the epithelial cells settled and formed a precipitate. We pour out the water, and add a lysis buffer (detergent) to the test tube to destroy the cell walls and heat it in a water bath.

    As a result, instead of whole cells, a smoothie appears in a test tube, like from a blender: the remains of membranes, the cytoplasm with proteins dissolved in it, cellular organelles and DNA. We remove 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 (as salt dissolves in water), and nonpolar molecules dissolve only in nonpolar liquids (essential oils - in alcohol).



    Therefore, we add a non-polar solvent chloroform to the test tube (DNA does not dissolve in it, and cell membranes - yes) and send it to centrifuge.
    As a result, we get a two-phase solution: water with a DNA film at the top and chloroform that has sunk to the bottom. It is necessary to carefully draw the aqueous part with DNA with a Pasteur pipette and transfer to another tube and pour chloroform. To the honor of geeks, we say that even the youngest guys ventured to carry out this jewelry operation.
    Then we added a sediment solution in order to once again wash the DNA from the detergents.
    DNA is a charged particle, so we added a salt solution with high ionic strength to precipitate it. (At some point, it ended with us, I had to run to the food court, take ordinary table salt and breed it). Toward the end - shock therapy: add icy 96% ethanol. Ten minutes in the freezer, ten thousand circles in a centrifuge - and we can see the DNA at the bottom of the tube.
    We drain the liquid, and the precipitate itself is dissolved in deionized water. Such water does not interfere with the functioning of enzymes that are used in molecular biology, so it can be used for PCR.

    Everything else took place in our laboratory tent (read - in a clean field).



    Using PCR, we obtained many copies of the site at the very end of our gene.
    Two primers determined the beginning and end of the DRD4 gene repeats. They cut it out and copied it many times using the enzyme polymerase, which reproduces the nucleotide sequence in the image and likeness of the originally existing gene segment. 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 samples obtained after PCR into the wells of the agarose gel.



    Then an electric current was passed through the gel. 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 length of DNA. We added a special solution to the gel, which highlighted the DNA if the gel was exposed to ultraviolet light on a transilluminator. The distance was measured with a molecular weight marker - and voila, you determined the adventurous "slow" 7R allele.



    The whole analysis took about two hours (excluding the process of DNA extraction).

    In just two days of the picnic, about 150 people passed genotyping, of which 13% were genetic adventurers. Moreover, on average for the Russian population, variant 7R is found in only 5% of people . It turns out that the concentration of genetic adventurers at HikPiknik increased.

    By the way, one of the adventurers on Saturday was so carried away by field genetics that he came to us on Sunday as a volunteer.

    UPD : in St. Petersburg, the concentration of genetic adventurers is lower: only 2.5%.
    In total, for the second weekend of Gikpiknik in St. Petersburg, we progenotyped 160 people.


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