Tartus geneetikuid mängimas

4.aprillil sai 15 meie kooli õpilast  võimaluse osaleda Tartus geneetika õppepäeval, mida korraldas Mari Järve.
 Siin  üks muljetus . Autor Oliver Valdmaa.

Genetics Learning Day

In Ahhaa Science Center

DNA is made up of deoxyribose, phosphoric acid and sequence of 4 different nucleotides. The sequence of nucleotides A, T, C, G is according to different evaluations 99% the same but there is a difference of about 1% of the DNA sequence, which enables to differentiate different people in forensics. That is why DNA samples are used to verify or identify people in forensics. DNA collected from the crime scene is compared with the DNA collected from the suspect. Not the entire DNA is compared because it would be too much data to analyze since one human DNA molecule consists of about 3 billion base pairs.

In Ahhaa we made an experiment of identifying the accused suspect so we compared three suspects’ DNA with the two DNA samples collected from the crime scene of raping. As a negative test we used water instead of DNA. Since the suspect can only be male, we are comparing the genetic markers on the Y-chromosome. We are looking for a one base substitution on the Y-chromosome. According to base substitutions men can be divided into haplogroups. There are many men in each haplogroup. So in our experiment we cannot be 100% sure that the suspect is committed the crime. Our experiment can still be used as a simulation though. We tested 3 haplogroups common in Estonia – N1c, R1a and I2a. Specifically our group tested haplogroup I2a, which has a mutation M423.

First of all in the experiment we had a small amount of all the DNAs. We had to reproduce the DNA in order to get more DNA, which would enable analyzing it. For that we used the polymerase chain reaction (PCR), which made copies of the specific part of the DNA we were interested in.

In the PCR, the two DNA chains were heated to the temperature of denaturation, which was 95 degrees Celsius. At about 40-60 degrees a primer containing up to 30 base pairs attaches to both chains, which were separated. DNA polymerase synthesized a new complementary chain to the previous chain and the DNA polymerase used dATP, dTTP, dGTP, dCTP which will be the nucleotides constructing the complementary chain. This cycle is done repeatedly in order to produce billions of DNA copies. Our first task was to mix the DNA, PCR mix containing dATP, dTTP, dGTP, dCTP and other necessary substances (had a green color), DNA primers and water. This mix was put into the PCR-machine with a working cycle of 1.5 hours. 

We used restriction enzymes, which “cut” DNA molecule in half only if there is a specific sequence of base pairs. Our sample DNA size was 370 base pair. If the sample had a mutation the restriction enzyme would not cut the DNA and if the enzyme did not have a mutation M423 then the restriction enzyme would cut the DNA sample between base pairs 207+163. The buffer was orange color. So we had to use a pipette to put the enzyme ssiI, with a concentration of 0.5 l and the rest of the 15l mix that we made for 10 samples was the orange buffer, into the DNA samples. The mix is put again into the PCR-machine that worked at the temperature of 33.5 degrees Celsius for 1.5 hours.

In order to find out whether the DNA had the mutation we were looking for (M423) we had to put the mix from the PCR on the specified holes on the agar gel. The gel is located in a special buffer mix bath, which will be electrified and lightened with the UV-light. This procedure is called gel-electrophoresis. DNA has a negative charge and it starts to move towards the positive pole. So we can see if the restriction enzyme cut the DNA. Agar gel contains ethidium bromide, which attached to DNA and glows in the UV light and we can see the glowing DNA in the UV light. Electrophoresis takes about 40 minutes.

From the experiment we made we could find out that the Suspect no. 2 is guilty of the crime since he belonged to the same haplogroup R1a as the DNA collected from the crime scene also belonged to R1a haplogroup. Sample 1 from the crime scene collected had M423 mutation but we could not confirm it with any of the suspects so in order to find out the criminal, our test did not give validation of who to charge with crime.

Overall, I found the experiment very interesting and well constructed and I gained new knowledge of how PCR works and how DNA tests are carried out. I did not know before that the DNA tests are actually so complicated and consume a lot of time.