16 December 2015

DNA in criminology

Gene Printing

Oleg Makarov, "Popular Mechanics" No. 9-2015
Published on the website "Elements" 

When in the XIX century it was found out that the papillary pattern on the fingertips is unique for each individual, fingerprinting revolutionized criminology. Left an imprint at the crime scene – you can't get away. The criminals armed themselves with gloves and napkins to erase the "fingers". But it became much more difficult for them when a new discovery was made: our individual "passport" is stored in each of the hundred trillion cells of the human body.

Yes, we are talking about DNA and molecular genetic examination. Everything seems to be simple: DNA is taken from the cells left by the criminal and compared with DNA from the cells of the suspect. In fact, of course, the simplicity here is apparent. If some living cells can be seen through a weak school microscope, or even with the naked eye (for example, an egg), then DNA is an object of the microcosm, and it will not be possible to "take" it so easily. The same with "comparison". Human DNA consists of 3.1 billion base pairs. Reading (sequencing) all this amount of information is an extremely time–consuming task.

How many cells do I need?We talked with representatives of Gordiz, the only Russian manufacturer of reagents for genetic forensic examinations, about how a unique genetic portrait of a person is compiled and what technologies are used in this case.

To begin with, I wanted to find out what minimum of biological material a criminal should leave at the crime scene in order for molecular genetic analysis to become possible.

"In principle, you can get a result from one cell," says Sergey Leonov, a biochemist who has worked as a forensic expert for several years. – There are, for example, devices – laser microdissectors that allow you to cut single cells from the samples under study. However, working with such a small amount of genetic material does not belong to standard technologies and processes and does not guarantee high reliability. For a normal process, at least 10-15 cells are needed, and in fact this is a very small amount. One touch of the hand to an object leaves hundreds of cells on it."

Yes, a person generously scatters genetic material, but already at the stage of searching for biological traces of the criminal, forensic experts are waiting for a lot of problems. For example, an excellent material for research is blood, traces of which are clearly visible and there is usually a lot of DNA in them. But the bloodstain could have mixed the blood of the perpetrator and the victim. How to separate the cells from each other? The task is somewhat simplified if the perpetrator and the victim are of different sexes: then preliminary sorting by the presence or absence of the Y chromosome is possible. In general, working with mixed objects is a common situation: as a rule, the objects that the attacker touched were touched by someone before him, and there you can find biomaterial belonging to dozens of people. To isolate the necessary material, a quantitative method is sometimes used: the trace of the criminal is the freshest, which means that if the number of some cells on the surface of the object is at least an order of magnitude higher than the number of others, we can assume that this is the desired biomaterial.

A typical case: after rape, the victim's epithelial cells are mixed with the perpetrator's sperm. A special method has been created for this case. Since the epithelium has less structural strength compared to male germ cells, it can be destroyed with special chemicals, leaving the spermatozoa intact and obtaining a pure fraction of the rapist's cells.

Thus, the actual molecular genetic research is preceded by the search for the necessary material and its isolation from different mixtures. There are, for example, chemical methods of searching for traces of blood where these traces were tried to be washed. There are technologies for finding cells left on paper and other materials.

DNA LaunderingAnother issue is the safety of the biomaterial.

On the one hand, the genome of a Neanderthal and a mammoth has been read, that is, DNA can be preserved for tens of thousands of years. And on the other hand, in the blood stain left by the criminal a couple of days ago, DNA may not be at all. How so? "DNA is one of the most stable biomolecules,– explains Sergey Leonov. – If there are conditions for storage, it can remain intact for an almost unlimited amount of time. The best conditions are drying, for example on paper. Personally, I had to participate in the work on the identification of the remains of soldiers who died during the Great Patriotic War. The DNA found in the remains was compared with preserved DNA samples from frontline letters provided by relatives of soldiers who died in these places."

DNA is rapidly destroyed by various biological, chemical and physical factors. If the biomaterial gets into a humid environment, rotting immediately begins. Rotting is nothing more than the eating of cells by bacteria. Bacteria also secrete special substances – nucleases, which destroy all the nearby gene material: it is a weapon of virus control. If a handkerchief with a blood stain has become an object of putrefactive processes, it may turn out that there is a blood stain, but there is no DNA in it anymore. The presence and concentration of the gene material in the samples will be detected by chemical methods during chemical analysis, but before this happens, it is necessary to extract DNA from the cellular environment. "To do this," says Sergey Leonov, "special reagents are used that completely destroy the cell material, leaving only DNA. The DNA is then electrostatically bound to the sorbent, which is extracted and then washed off with water. So it turns out a pure aqueous solution of DNA." If, according to further analysis, the genetic material is in solution and in the required amount, you can start creating a genetic "fingerprint".

The benefits of silenceFirst of all, it should be noted that there is no need to compare the two DNA completely.

Genetically, all people on Earth are 99.99% identical, and therefore it makes sense to analyze only the similarity-the difference of certain parts of the genome that have polymorphism, that is, they are represented in different people in several different species. The "gold standard" today is the study of polymorphic markers with variable length. Markers are sections of "silent" DNA that do not act as active genes and do not encode proteins. But they have a characteristic structure – the same sequences of base pairs are repeated in them several times. A different number of repetitions forms the same polymorphism. Of course, the number of variants of the length of one marker is not equal to the number of people on Earth. If a marker has (conditionally) ten options, then having a marker of a certain length means only belonging to one of the ten groups of humanity, numbering hundreds of millions of people each. But if we add data on the second polymorphic marker here, the group where such a combination of lengths occurs will noticeably narrow. It will be about millions of people. When adding new markers, as they say in science, the discriminatory potential will exponentially increase, and finally, the number of markers can be brought to such a number that the combination of data on their lengths will become unique for an individual, and the probability of repeating this "pattern" will practically be reduced to zero. The key to identity identification is just a dozen polymorphic markers. But then we remember that DNA is an object of the microcosm, and it is impossible to measure its sections with a ruler or a caliper. Biochemistry and electricity come to the rescue here.

Stage one. The Great multiplication of markersThe scheme of the polymerase chain reaction shows the main stages of multiple copying of the DNA site of interest (marker).
At the denaturation stage, under the influence of heating, the DNA chain splits into two strands. At the annealing stage, during cooling, labels-primers are attached to individual DNA strands. At the elongation stage, the polymerase enzyme completes the second strand to the site under study.Constructive broth

Molecules, even as complex as DNA strands, are so small that molecular genetic research requires not one copy of the genome region of interest (marker), and not ten, or a hundred, but millions and billions.

Thus, a significant concentration of marker copies is required, which will make it possible to physically indicate the parameters. The core, the most difficult moment of the entire identity identification technology is polymerase chain reaction (PCR), which just allows you to accumulate as many markers as you need. The reaction takes place in a solution where the DNA under study is present, peculiar labels-primers (actually structurally identical to small sections of one DNA strand), free–floating nitrogenous bases forming DNA - adenine (A), guanine (G), cytosine (C), thymine (T), catalysts and, finally, the main acting character is the enzyme Taq polymerase. The reaction occurs under variable temperature conditions. First, with the help of heating, DNA is denatured, that is, its "double helix" splits into two strands, and the base pairs open. Then, when cooled, the primers mark the boundaries of the marker we are interested in. They are adjacent to the DNA strands, in a small area, as if restoring the double structure of the molecule. After that, the polymerase "takes" nitrogenous bases from the solution and, working on one side of the marker, completes the second strand for each of the strands of denatured DNA. As a result, instead of one DNA, two molecules with a partially restored double structure appear in the solution (in particular, for a marker). Then everything repeats: denaturation with separation of two strands, primers and polymerase work. As a result of the chain reaction, we get a huge number of copies of a marker having a normal double structure.

Stage two. Making up a genetic portraitThe diagram shows the classical scheme of genetic analysis using agarose gel.
Markers move under the influence of an electric field in the gel block. Since smaller markers meet less gel resistance, they break out ahead. The final identification of markers takes place with the help of laser illumination, which excites fluorescence."PCR is a very complex and expensive process," says Vladimir Orekhov, CEO of Gordiz.

– The number of companies in the world that produce reagents for such a reaction can be counted on the fingers of one hand, and one of them – ours – is located in Russia. The main difficulty is that all markers should be copied in the test tube at the same time and not interfere with each other. How many markers do I need? The American CODIS standard includes 13, the European ESS – 12. Although the markers from the two systems overlap, there are differences. To meet the needs of specialists working within different standards, we produce reagents for PCR with the participation of 19 markers. This amount will be enough to identify a unique person, even if the world's population grows by several orders of magnitude. We also have our own know-how. The fact is that usually PCR reagents are produced in liquid form, which imposes special requirements on storage and transportation conditions (in particular, freezing is needed). We produce reagents by drying raw mixtures, and they do not need any special conditions for transportation. All this reduces the cost of the process and simplifies the task of delivering reagents, for example, to remote areas. I note that all the raw materials for reagents are of domestic production."

Racing in the fieldThe PCR product – a solution with a high concentration of markers – is a ready-made base for measurements.

The methods may differ slightly, but they are based on a process familiar to many who have visited physiotherapy rooms in polyclinics. This is electrophoresis – the movement of charged molecules in an electric field.

"Until now, in Russia, the method of separating the PCR product on polyacrylamide gel is used," says Sergey Leonov. – Genetic analyzers have replaced this method, which dates back to the 1980s. In them, the movement of molecules occurs inside the capillaries of a porous polymer. But the essence is the same: the marker, being a cation, begins to move when an electric field is applied, however, due to the resistance of the medium, the longer molecule slows down, and the shorter one moves faster."

A genetic analyzer is a device that represents a more advanced technology for sorting markers isolated from DNA. Here the markers move in capillaries made of a porous polymer. The photo shows the device "NANOPHOR-05", produced by the Russian company "Syntol"

Thus, the molecules are separated by length. However, some markers may have the same length (the same number of base pairs), while differing in the structure of the placement of bases. To avoid confusion, potentially similar-sized markers receive a fluorescent label (one of four or five colors) during the PCR process. In a genetic analyzer, markers are illuminated by a laser, the beam of which excites a glow of a certain color. After that, the token can be considered definitively identified. The final document of the analyzer is a table in which the presence of certain variants (alleles) of all the markers studied in the genome is marked in color. Is that all?

Without witnesses"No, not everything," says Vladimir Orekhov.

– If the samples of the trace and living tissue match, the question of statistics arises – how likely is a coincidence. Theoretically, the probability exists, and according to the results of molecular genetic studies, the probability of 100% is not given. Another thing is that the probability of accurate identification is 99% plus a lot of digits after the decimal point. But in order for the conclusion to have all the signs of scientific validity, this probability must be calculated mathematically, taking into account, for example, the population frequencies for each trait (a common trait is more likely to match, a rare one is less). In this regard, there is a myth that some types of forensic examinations give an unambiguous answer "yes" or "no", and genetic examination gives only a quantitative assessment of probability. But in fact, it is genetics that gives the most accurate answer, and, for example, handwriting or fingerprinting can be wrong, and such cases are known. Of course, the human factor may interfere with the genetic examination (the laboratory assistant mixed up the sample, etc.), but if the study is carried out correctly, it gives a strictly scientific and accurate result."

Over time, molecular genetic examination has become an increasingly common and less expensive method of criminology. At the same time, its effectiveness is extremely high. Since the 1990s, laws on genomic registration have been in force in the USA, Germany, and the UK. According to these regulations, biological material left at the crime scene by unidentified persons is examined and entered into the database. The gene "passport" of people serving sentences for particularly serious crimes is entered into the same database. Such a database turned out to be a very effective means of solving crimes in which there are no suspects or witnesses, since in a large number of cases unidentified genetic material sooner or later found its owner through the database or showed a connection with other crimes committed. Such a law was adopted in 2009 in Russia, but its financing began only recently, and it is premature to talk about a serious effect for our country.

Portal "Eternal youth" http://vechnayamolodost.ru
Found a typo? Select it and press ctrl + enter Print version