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The article is devoted to the development and use of a method for diagnosing the source of Brachyspina Syndrome, or short spine, in the Holstein breed and its crossbreeds. Representatives of this breed are the most highly productive animals in the world for milk. The development of a diagnostic method for breed-specific hereditary carriers is an important task in dairy farming. In this regard, the authors have proposed a Patent and a Reagent Kit for the detection of normal FANC1TY and mutant FANC1BY alleles in the Holstein breed and its crossbreeds. The frequency of occurrence of genotypes formed by these alleles in different sex and age groups of animals in the Holstein breed and its high-blooded hybrids was studied. Historical data related to the founders, from whom the spread of the mutant FANC1BY allele within the Holstein breed itself and its crossis in the USA, Canada and Russia began, has presented. The development of a diagnostic method for the mutant FANC1BY allele will make it possible to stop spreading of the Brachyspina Syndrome source. The proposed method will make it possible, in the early stages, to form healthy groups of breeding animals (bulls, replacement bulls, bull-reproducing cows), thereby laying the foundation for creating a high-quality livestock in the regions where the Holstein breed and its numerous crossbreeds are bred. cattle, Holstein breed, crossis, genotype, FANC1BY mutation, allele, Brachyspina syndrome

In forensic medicine, it is necessary to establish the sexual identity of the owner of the analyzed biological material. To do this, it is necessary to use PCR to detect specific DNA sequences that are characteristic only of the Y chromosome. For these purposes, a number of loci are used, located on both the Y and X chromosomes but carrying certain differences in the nucleotide sequences (alpha satellites DYZ and DXZ; amelogenin loci AMELY and AMELX; STS steroid sulfatase genes; the genes of the neuroligin NLG4Y and NLG4X, etc.), and those located only on the Y-chromosome (sex-determining region SRY; gene of the specific testicular protein TSPY, etc.). At the same time, forensic experts often deal with damaged or old samples in which the DNA has been destroyed and extended fragments in it may simply not be, as a result of which false negative results will be formed. Thus, in DNA forensics, when detecting gender loci, the sizes of amplicons should tend to the minimum possible. Therefore, in this review article, a certain emphasis was placed on the size of amplicons, and as practice shows, for most loci, their minimization is in demand. Moreover, such a PCR analysis in a number of cases (in XX-men, XY-women, in persons with other sex chromosome abnormalities, in people who deliberately changed their gender identity) it can lead to a false definition of the phenotypic sex due to the genetic characteristics of such individuals. As a result, the ongoing investigation of a crime, focused on the search for a representative of a particular gender, can go down the wrong path. A cardinal solution to this problem in DNA criminology can be a universal DNA registration of the entire population, which will allow for the biological traces with high accuracy to establish a specific person to whom these traces belong and his real sex will no longer be important and it will not be relevant to determine it with the help of PCR. In addition to forensic medicine, the problem of establishing gender also exists in women's sports. For a whole decade, the PCR method and some of the loci listed above were used for this purpose, but since 2011 PCR has been abandoned and instead the level of the male hormone testosterone has became determined. However, with the gender of female athletes, there are much more ethical issues than genetic ones.

The processes occurring during the enzymatic growth of the DNA chain in the form of elongation of the molecules, the release of pyrophosphate, proton, thermal energy, and an increase in electrical impedance, which are used in various methods of high-throughput DNA sequencing by synthesis, are briefly considered. The detection of DNA chain growth is controlled by high-voltage gel electrophoresis and has limited scalability. As for mentioned above other by-products of DNA chain polymerization, their detection can be easily scalable, which has led to the emergence of methods for whole genome sequencing of new generations of DNA, which have received the widely used abbreviation NGS - Next Generation Sequencing. However, the attribution of any new sequencing method to a particular generation is sometimes difficult due to the fact that the principle used in it was born earlier than the other one was implemented, which turned out to be less productive in the end. In addition, it is more important to distinguish the methods of new DNA sequencing into two groups in which the massive parallel sequencing of identical matrices takes place or the sequencing of single DNA molecules takes place and last one have received the designation monomolecular sequencing. In this review, along with the classical Sanger method of DNA sequencing, which is still the "gold standard", pyrosequencing, semiconductor sequencing, thermosequencing, electronic sequencing, fluorescent bridge sequencing and sequencing using nanoballs from the first group, as well as monomolecular methods – tSMS sequencing, SMRT sequencing and nanopore sequencing are considered. Attention is paid to the costs of DNA sequencing and the prospects for its development.

A new coronavirus infection in two years of the pandemic led to the fact that the number of individual SARS-CoV-2 virions formed in patients and in asymptomatic carriers exceeded a sextillion (1021) and the law of the dialectic of the transition of quantity to quality in the form of RNA polymorphism of this quasispecies inevitably had to come into effect. At the same time, virions differing in nucleotide sequences may simultaneously be present in individuals. Most likely, the diversity of coronaviruses is growing due to people with weak immunity, in whom SARS-CoV-2 persists for a long time, undergoing mutations caused by both the action of vaccines and drugs, which eventually generates the appearance of dangerous variants of the virus that contribute to the emergence of pandemic waves. There are five such Variant of Concern of coronaviruses, to which the World Health Organization recommended assigning designations using letters of the Greek alphabet, so far – Alpha, Beta, Gamma, Delta and Omicron, and the latter, having appeared quite recently, has already managed to practically displace all previous variants. Hypotheses trying to explain the unexpected appearance of an Omicron that has accumulated a very large number of mutations are considered. One of the most plausible versions is that it assumes the imperceptible evolution of this coronavirus, due to the fact that at first it did not pose any threat and did not come to the attention of specialists, but after one or two final mutations, it acquired a fundamentally different ability to multiply and massively infect people. There is no clear answer yet whether the letters of the Greek alphabet will be enough to designate new variants of SARS-CoV-2. But in any case, despite the escape of Omicron (and possibly new variants, including its derivatives) from protective antibodies, vaccination is the protection against COVID-19, since even without being able to resist the infection itself, they make it difficult for the virus to multiply inside the human body and thereby facilitate the course of the disease. However, it is necessary to create new vaccines taking into account mutated variants.

The features of the nuclear genome of dogs, including a number of reference ones, are briefly considered, paying the greatest attention to single-nucleotide DNA polymorphism in the form of so-called SNPs. It is noted that the detection of SNPs implies two processes in the form of their de novo detection by DNA sequencing, including whole genome sequencing, as well as the detection of already known SNPs in genomes during replicative studies on the large experimental material. Some attention is paid to specialized databases and web-resources on canine SNPs. The issues of DNA registration of individual dogs on the basis of single-nucleotide polymorphism are touched upon, which can have various applications, including forensic purposes and for dog control, contributing to the elimination of stray dogs as a class.

The analysis of DNA fragments by capillary gel electrophoresis is in wide current used in the practice of research and testing laboratories primarily for highly accurate and highly sensitive identification of organisms by their DNA traces separated from biological test materials and amplified by polymerase chain reaction. A constantly growing flow of examinations performed using this method necessitates the optimization of research protocols in order to shorten the analysis time without loss of separation efficiency. The effect of electric field voltage on the separation efficiency of single-stranded DNA fragments in a length range from 60 to 1200 nucleotides in a series of polymers based on linear poly(N,N-dimethylacrylamide) was studied. The best values of electric field voltage were experimentally determined to be recommended for DNA fragment analysis on the first Russian genetic analyzer Nanophore 05 with the studied polymers.

В 2020 году исполнилось 100 лет термину «геном», предложенному немецким ботаником Г.Винклером при описании партеногенеза в растительном и животном царствах. Этимология данного слова неясна, но нам представляется, что Винклер использовал игру слов, соединив морфемы «ген», «сома» и получив “genosom” (нем.), после чего решил удалить один слог, что и привело к краткому и емкому слову «геном», объединяющему гены и составляющем для них нечто целое, что также совпало с окончаниями «–ом» отдельных биологических терминов, обозначающих некое множество (в данном случае для генома – генов). Первоначально под геномом подразумевался гаплоидный набор хромосом, и таким образом хромосомы служили элементарной единицей генома. Сейчас же с появлением возможности секвенировать полный геном его элементарной единицей стала пара нуклеотидов, но и хромосомы оказались задействованными, поскольку нуклеотидные последовательности распределяются в базах данных по хромосомам, когда такое по результатам полногеномного секвенирования становится возможным. На протяжении столетия менялись взгляды ученых, что считать геномом, подразумевая под этим его гаплоидную или диплоидную природу. Сейчас принято считать, что геном - это нуклеотидные последовательности всей совокупности генов и прочих участков ДНК гаплоидного набора хромосом конкретного вида организмов, однако и габитус и функциональное состояние каждого организма определяется его полным диплоидным геномом, тогда как при сборке гаплоидного генома a priori происходит игнорирование тех или иных азотистых оснований в отличающихся аллелях, поскольку выбирается лишь один какой-то нуклеотид, что сразу снижает ценность такого генома, которая все равно очень высока, поскольку секвенирование полных гаплоидных геномов уже очень большого числа видов организмов различного уровня генетической сложности дало весьма важную информацию о Живом. Тем не менее, для персонифицированной медицины будущего нужны знания об исключительно диплоидных геномах людей, получение которых пока представляет серьезную проблему, в отличие от нынешних квази-гаплоидных геномов, которые секвенируются почти массово. Диплоидные геномы растений также представляют интерес, в том числе для CRISPR/Cas геномного редактирования, когда необходимо произвести изменения в обоих аллелях парных хромосом, и для этого знать их возможно отличающиеся нуклеотидные последовательности крайне необходимо. Равно как и по завершении такого редактирования следует с помощью полногеномного диплоидного секвенирования выявить произведенные целевые и нецелевые мутации. Разрабатываемые подходы полногеномного секвенирования, в том числе протяженных участков ДНК вкупе с компьютерными программами, рассчитанными на фазировку данных, недавно вызвали к жизни новый термин «гаплотиг», представляющий собой гаплотип-специфичный контиг. Вне всякого сомнения, все это позволит со временем перейти к уверенному установлению полных диплоидных геномов, что придаст термину «геном» новый смысл, считая его для эукариотических организмов диплоидным, что будет к тому же отражать и саму сущность организации генома, имеющего в норме двуродительскую природу.

Specific amplification of nucleic acids is a convenient and quick alternative to the culture-based method of detecting bacterial cells. However, conventional PCR and other amplification reactions can not differentiate between live bacteria and dead or dormant ones, and are also capable of amplifying DNA that persists for a long time and in a cell-free state. Several methods have been developed in order to establish the viability of microorganisms by amplification of specific sequences of nucleic acids, both those controlled by changing temperatures and isothermal ones. For some of them, DNA modified by phenanthridine dyes serves as a target, and simultaneous use of monoazides of ethidium and propidium was shown to be preferable for the purpose. For other methods, the targets are directly RNA molecules or their cDNA copies. Pre-rRNA detection seems to be the most preferable approach, due to the presence of these types of RNA exclusively in living cells.

The paper considers the problematic aspects of detecting a new coronavirus infection using RT-PCR, which often lead to false negative diagnostic results, which occur both at the preanalytic stage and during nucleic acid amplification, including the interpretation of the obtained data. The viral load in humans was evaluated and assumptions were made about the expected number of viral particles in the studied oropharyngeal and nasopharyngeal samples. The list of diagnostic test systems approved for use in the Russian Federation for detecting SARS-CoV-2 with their brief characteristics is given. The necessity of simultaneous use of several targets in the coronavirus genome in one test system detected by probes with the same fluorochrome is noted, which on the one hand increases the probability of detection by increasing the signal, and on the other hand eliminates the false negative results that could occur in the case of mutations in the virus genome at the sites of annealing primers and hybridization probes.

The quantitative trait loci associated with the immune properties of chickens are of interest from the point of view of obtaining animals resistant to infectious agents using marker-assisted selection. In the process of selecting markers for genomic selection in broiler-type chickens, a non-standard genotype frequency of the RACK1 gene allele (SNP Gga_rs15788101) in the B5 line of broiler-type chicken cross Smena 8 was identified and it was suggested that this gene was involved in selection. Therefore, it was decided to investigate the available polymorphisms in the three genes responsible for the IgY titer (DMA, RACK1 and CD1B). Molecular typing of single nucleotide polymorphisms of three loci revealed an approach to fixation of the unfavorable allele of the DMA gene (SNP Gga_rs15788237), an approach to fixation of the unfavorable allele of the RACK1 gene and the prevalence of the favorable CD1B gene allele (SNP Gga_rs16057130). Analysis of the haplotypes revealed a strong linkage disequilibrium of these genes. This suggests that these genes experience selection pressure. Analysis of the protein-coding sequences of the CD1B and DMA genes of various breeds of chickens revealed a negative selection of these genes. In order to understand whether the fixation of the studied alleles is the result of artificial selection of the B5 line of the cross Smena 8, an analysis of similar loci in layer chickens Hisex White was carried out. The frequencies of the alleles at the loci of the CD1B gene (Gga_rs16057130) and the RACK1 gene (Gga_rs15788101) in the Hisex White chicken genome differ from the frequencies of the alleles obtained for chickens of the B5 line of the cross Smena 8. It can be assumed that the fixation of the allele in the DMA gene (SNP Gga_rs15723) is associated with artificial or natural selection, consistent in broilers and layers. Changes in the loci Gga_rs16057130 and Gga_rs15788101 in the B5 line of the Smena 8 chickens are most likely associated with artificial selection of broiler productivity traits, which can subsequently lead to fixation of alleles at these loci. Artificial breeding of chickens leads to degradation of the variability of genes encoding elements of the immune system, which can cause a decrease in resistance to various diseases. The study of the negative impact of selection of economic traits on immunity should provide means to mitigate negative consequences and help find ways to obtain disease-resistant animals.Локусы количественных признаков, связанные с иммунными свойствами кур, представляют интерес с точки зрения получения устойчивых к инфекционным агентам животных при помощи маркер- опосредованной (геномной) селекции. В процессе подбора маркеров для геномной селекции у кур брой- лерного типа выявлена нестандартная частота аллеля гена RACK1 (SNP Gga_rs15788101) у кур линии Б5 мясного кросса селекции СГЦ «Смена» и возникло предположение о том, что этот ген вовлечен в селекцию. Поэтому было решено исследовать доступные полиморфизмы в трех генах, ответственных за титр IgY (DMA, RACK1 и CD1B). Молекулярное типирование однонуклеотидных полиморфизмов трех локусов показало при- ближение к фиксации неблагоприятных аллелей генов DMA (SNP Gga_rs15788237) и RACK1 и преобладание благоприятного аллеля гена CD1B (SNP Gga_rs16057130). При анализе гаплотипов выявлено сильное не- равновесное сцепление этих генов. Это свидетельствует о том, что данные гены испытывают селекционное давление. Исследование белок-кодирующих последовательностей генов CD1B и DMA различных пород кур продемонстрировало негативную селекцию этих генов. Для того чтобы понять, является ли фиксация изученных аллелей результатом направленной селекции кур линии Б5 мясного кросса СГЦ «Смена», проведен анализ аналогичных локусов у кур яичной селекции «Хайсекс белый». Частоты аллелей в локусах гена CD1B (Gga_rs16057130) и гена RACK1 (Gga_rs15788101) в геноме кур «Хайсекс белый» отличаются от частот аллелей, полученных для кур линии Б5 мясного кросса селекции СГЦ «Смена». Можно предположить, что фиксация аллеля в гене DMA (SNP Gga_rs15788237) связана с искусственным или естественным отбором, единым для кур мясной и яичной селекции. Изменения в локусах Gga_rs16057130 и Gga_rs15788101 у кур линии Б5 мяс- ного кросса селекции СГЦ «Смена», скорее всего, связаны с искусственной селекцией признаков продуктив- ности бройлеров, которая в дальнейшем может привести к фиксации аллелей в этих локусах. Искусственная селекция кур ведет к деградации вариабельности генов, кодирующих элементы иммунной системы и, как следствие, уменьшению резистентности к различным заболеваниям. Изучение негативного влияния селек- ции хозяйственных признаков на иммунитет должно способствовать снижению отрицательных последствий и поиску способов получения резистентных к заболеваниям животных.

Цель исследования оценка эффективности разработанной нами диагностической панели, основанной на технологиях секвенирования следующего поколения (Next Generation Sequencing, NGS), для диагностики широкого спектра социально значимых наследственных дегенеративных заболеваний мозга. Материалы и методы. С помощью разработанной диагностической таргетной NGS-панели (на платформе Illumina MiSeq, США), которая направлена на секвенирование кодирующей области 300 генов нейродегенеративных заболеваний, проявляющихся двигательными и когнитивными расстройствами, проведен мутационный скрининг ДНК, полученной от 32 пациентов с неуточненным диагнозом. Результаты. Применение оригинальной генетической панели позволило выявить ряд редких форм наследственной нейродегенеративной патологии с идентифицированными (с помощью NGS) и подтвержденными (с помощью прямого секвенирования) мутациями в генах: спиноцеребеллярных атаксий и параплегий, паркинсонизма, дистоний, нейрометаболических заболеваний. У 11 пациентов выявлено 12 мутаций в 10 различных генах, обусловливающих развитие трех аутосомно-рецессивных заболеваний (гены DDHD1, NPC1 и RARS) и восьми заболеваний, связанных с аутосомно-доминантным наследованием (гены SPAST, SPTBN2, GRN, GCH1, LRRK2, NOTCH3 и AGER). Заключение. Панельный скрининг неясных случаев нейродегенеративных заболеваний, выполненный с помощью оригинальной таргетной модели, позволяет выявить и подтвердить последующие мутации в различных генах для более чем трети обследованных случаев.The aim of the study was to assess the efficacy of a diagnostic panel based on next-generation sequencing (NGS) and developed by our team, to diagnose a wide range of socially significant hereditary degenerative diseases of the brain. Materials and Methods. Using the diagnostic target NGS panel (powered by Illumina MiSeq, USA), designed to sequence the encoding area of 300 genes related to neurodegenerative diseases manifesting with movement and cognitive disorders, we performed a mutation screening of the DNA of 32 patients with an otherwise obscure diagnosis. Results. The application of the original genetic panel revealed a number of rare hereditary neurodegenerative pathologies with mutations in genes for spinocerebellar ataxias and paraplegias, parkinsonism, dystonias, and neurometabolic diseases, identified by NGS and confirmed by direct sequencing. In 11 patients, 12 mutations were found in 10 different genes, causing the development of three autosomal recessive diseases (genes DDHD1, NPC1 and RARS) and eight diseases associated with autosomal dominant inheritance (genes SPAST, SPTBN2, GRN, GCH1, LRRK2, NOTCH3 and AGER). Conclusion. Panel screening of uncertain cases of neurodegenerative diseases using this bespoke target model enabled us to reveal and subsequently confirm mutations in various genes for more than a quarter of the examined patients.

State-of-the-art microfluidic analytical systems are briefly surveyed. Attention is focused on the use of microchip capillary electrophoresis. The main results obtained in the development of a prototype analytical system with a laser-induced fluorescence detector for electrophoresis on a glass microfluidic chip are presented. Experimental data on electroosmotic flow and the distribution of sample fluorescence intensity over the cross section of a microchannel are analyzed. A procedure for the rapid analysis of oligonucleotides on a microfluidic chip is described.