Application Of Molecular Genetic Methods Research Articles

DNA ISOLATION FROM BIOLOGICAL MATERIAL OF MARALS

The main task of maral breeding isproduction increase of antlers. Application of molecular genetic methods in maral breeding contributes to increase in reliability and accuracy of assessment of productivity and breeding value of stag deer at an early age. The purpose of the research is to test methods DNA isolation from biological material of marals. The biomaterial was selected fromstag deer of Altai-Sayan breed (blood, antler crumbs, cartilaginous tissue of the auricles). The work on DNA isolation from maral tissue samples was carried out in the bioengineering laboratory at Altai State University (Barnaul). DNA isolation was conducted using the method based on application of Chelex-100TM Resin chelating agent (Bio-Rad, USA), the method based on DNA precipitation Diamond DNA (OOO ABT, Russia) and commercial kit based on magnetic particles AMPure XP (Beckman Coulter, USA). The purification degree of the isolated DNA was assessed by PCR efficiency in reactions of amplification of the genes of cytochrome oxidase 1, cytochrome B of mitochondrial DNA. The highest concentration of maral DNA was established in isolation based on DNA precipitation (Diamond DNA). The concentration of DNA in the solution was 13.20 ng/µl (blood), 11.30 ng/µl (cartilaginous tissue), 5.74 ng/µl (antler crumbs).

Environmental paleomicrobiology: using DNA preserved in aquatic sediments to its full potential.

SummaryIn‐depth knowledge about spatial and temporal variation in microbial diversity and function is needed for a better understanding of ecological and evolutionary responses to global change. In particular, the study of microbial ancient DNA preserved in sediment archives from lakes and oceans can help us to evaluate the responses of aquatic microbes in the past and make predictions about future biodiversity change in those ecosystems. Recent advances in molecular genetic methods applied to the analysis of historically deposited DNA in sediments have not only allowed the taxonomic identification of past aquatic microbial communities but also enabled tracing their evolution and adaptation to episodic disturbances and gradual environmental change. Nevertheless, some challenges remain for scientists to take full advantage of the rapidly developing field of paleo‐genetics, including the limited ability to detect rare taxa and reconstruct complete genomes for evolutionary studies. Here, we provide a brief review of some of the recent advances in the field of environmental paleomicrobiology and discuss remaining challenges related to the application of molecular genetic methods to study microbial diversity, ecology, and evolution in sediment archives. We anticipate that, in the near future, environmental paleomicrobiology will shed new light on the processes of microbial genome evolution and microbial ecosystem responses to quaternary environmental changes at an unprecedented level of detail. This information can, for example, aid geological reconstructions of biogeochemical cycles and predict ecosystem responses to environmental perturbations, including in the context of human‐induced global changes.

Molecular genetic methods in biomedical research. Part III: human gene diagnostics in clinical practice

Application of molecular genetic methods in the diagnosis and treatment of human diseases is extremely wide due to a huge amount of hereditary information contained in the human genome. Gene diagnostics allows establishing predisposition to diseases, identification of genetic abnormalities and prediction of pathological outcomes. In addition, gene diagnostics also enables prediction of the individual response to treatment in order to achieve the maximum therapeutic effect. Among all molecular genetic methods, polymerase chain reaction (PCR) diagnostics is a leading approach. Technical simplicity, low cost, high sensitivity and reliability of the method have made PCR diagnostics a routine modality for the risk assessment, diagnostics, and monitoring of the treatment efficiency. Here, we consider the application of PCR diagnostics for the abovementioned tasks and talk about the real-life examples of detecting mutations and chromosomal aberrations which may cause a disease. Further, we discuss the prospects of using a semi-quantitative PCR in medical practice and focus on pharmacogenetics as a key component of a personalised therapy. The lecture is aimed primarily at biomedical students and physicians and represents a continuation of the previous lectures published in Fundamental and Clinical Medicin.

Polymorphism of the gene GDF9 in sheep of Prikatun type of Altai Mountains breedand its correlation with indices of meat rate productivity

In recent decades in many countries of the world the development of sheep farming is determined by the efficiency of mutton production. One of the promising areas of selection improvement of sheep breeds of meat and meat & wool productivity is an application of molecular-genetic methods. To obtain high-quality mutton it is advisable to use the Altai Mountains sheep breed, which features by high energy of growth of young animals and their adaptability to all-year-round pasture management. One of the candidate genes responsible for sheep meat productivity is the gene of growth differentiation factor -GDF9 (growth differentia­tion factor 9). The article presents the results of research of polymorphism of the gene GDF9 and its relation with the parameters of meat productivity in young sheep of Altai Mountains breed. It was found that the rams-carriers of AA genotype outperformed their peers of AG and GG genotypes in terms of pre-slaughter weight, carcass weight, meat yield and meat content ratio by 1.62 and 7.01 kg, 1.34 and 3.98 kg (P<0.05), 1.21 and 1.86 abs. percent, 0.16 and 0.39 units (P<0.05) accordingly. The muscle tissue of rams of the desired genotype featured the highest content of protein, fat and, accordingly, energy value in comparison with AG and GG genotypes. The animals of AA genotype had the largest area of a rib eye and diameter of muscle fibers. The superiority over other genotypes achieved 1.55 cm 2 and 1.98 microns in average. In addition the higher number of inter-fiber and inter-bundle inclusions of fat and lower content of connective tissue stipulated higher marbling score (MB)-higher by 3.32 points. The obtained data in­dicate that it is promising methods to increase the number of homozygotic animals in term of gene GDF9 by intended selection of parent animals whose genotype contains a desirable element for further improvement of quantitative and qualitative parameters of meat rate productivity in sheep of Altai Mountains breed.

The scientific revolution in microbiology and its importance for practice

The accumulation of knowledge in the process of scientific and technological development does not occur linearly, but in leaps and bounds: periods of relative stagnation are replaced by scientific revolutions. The scientific revolution is the change of explanatory paradigms by the scientific community. A few years ago, the establishment of the taxonomic affiliation of microorganisms by cultivation and counting by methods of classical microbiology was fraught with a number of insurmountable difficulties. The complex composition of the media, the presence of an excess of inhibiting compounds in the composition of the nutrient media, the need to maintain extreme temperatures, the composition of gases and pressure for cultivation, the susceptibility of the culture to the effects of oxygen excluded uncultured microorganisms from the field of view of the researcher. The emergence of molecular genetic methods made it possible to study the diversity of microorganisms, bypassing the previously obligatory stage of cultivation, isolation of a pure culture and comparison with a reference sample. Through the prism of scientific revolutions and changes in scientific paradigms, the differences between the methods of classical microbiology and molecular genetic methods are revealed. The experience of BIOTROF LLC in the application of molecular genetic methods for the identification of microorganisms in livestock and poultry farming is summarized. Based on the use of 16S metagenomics, it was possible to show for the first time that health disorders of farm animals and birds, a decrease in productivity and the duration of economic use in many cases are associated with dysbiotic phenomena in the gastrointestinal tract. Thus, in cows culled due to lactic acidosis, in comparison with clinically healthy animals, inhibition of the growth of bacteria synthesizing cellulases and microorganisms of the order Selenomonadales, capable of fermenting lactic acid to volatile fatty acids, was observed. Based on the calculation of Pearson's correlations, it was shown that an increase in the content of lactobacilli of the family Lactobacillaceae in the intestines of poultry was associated with an increase in meat productivity, and an increase in staphylococci, with a decrease.

Introduction of Innovative International Experience of Molecular Genetic Expertise in Ukraine: Legal Aspect

The article highlights the legal aspect of introducing innovative international experience of molecular genetic expertise in Ukraine. The authors provide specific proposals for regulatory regulation of such examinations. The authors conclude that genetic analysis is one of the most modern, effective and indispensable for crime detection and law enforcement in general. The practical application of genetic analysis with predictable and positive results is not possible without effective legal regulation that responds to current challenges and technologies.The purpose of the article is to investigate the legal aspect of introducing innovative international experience of molecular genetic expertise in Ukraine, to identify ways to improve legal regulation.Materials and Methods: The study material is a modern international regulatory framework that defines the principles and procedure for the application of molecular genetic methods. Methods of searching, analyzing, organizing, and summarizing information were used in writing the article.Molecular genetic studies are becoming more widespread and widely used. At the same time, the use of such technologies requires a proper settlement of ethical and legal issues.Genetic analysis is one of the most modern, effective and indispensable methods for crime detection and law enforcement. The practical application of genetic analysis with predicted and positive results is impossible without effective legal regulation that has to meet current challenges and technologies. The current legal and regulatory support for expertise in Ukraine, especially molecular genetic ones, does not meet current requirements and does not meet the needs of justice. Improving Ukrainian law requires a comprehensive approach, and adopting a separate law is one aspect of such improvement..

Applying Principles of Multi-Factor Genetic Analysis of Infectious Disease Agents in the Work of the Rospotrebnadzor SAET during Mass Events

Objective of the study – to develop and test in operation of the Rospotrebnadzor Spesialized Anti-Epidemic Teams (SAET), deployed during the major mass events, the procedure for application of molecular-genetic methods for detection and identification of pathogenic biological agents. Materials and methods. Utilized are the reporting documentation drawn up by the results of work of the specialized anti-epidemic teams of the Rospotrebnadzor on the provision of sanitary epidemiological welfare during Olympic Games 2014 and other important mass events in Sochi in 2015–2017. Results and conclusions. Developed and tested in practice of the Rospotrebnadzor SAET during the major mass events has been procedure for application of molecular-genetic methods, based on structured approach (algorithm) of indication and genetic characterization of PBA. Equipping of the SAET with diagnostic preparations is carried out in accordance with the provision of preparedness for performing investigations by three-level algorithm of molecular-genetic characteristics of pathogens: level I – detection (indication) of a pathogen using PCR, level II – identification of the pathogen genome fragments using PCR, level III – genotyping. Developed procedure was used successfully by the SAET during the Olympic Games 2014 and other important mass events in Sochi in 2015–2017, which allows for recommending it for future use.

The characteristic of diarrheagenic Escherichia separated from children aged under 5 years old in Yaroslavl.

The diarrheagenic bacteria coli take a significant place among agents of acute intestinal infections in children aged under 5 years. The main danger among these pathogens is represented by both enterotoxigenic E. coli causing enteritis and enterocolitis accompanied by acute dehydration diarrhea and Escherichia producing shiga-toxin being agents of hemorrhagic colitis and hemolytic uremic syndrome. The fast and proper identification of agents of these two groups of pathogens is an important task of bacteriologists to be resolved for successful treatment of patient because tactics of therapy of enterotoxigenic diarrhea and hemorrhagic colitis and hemolytic uremic syndrome significantly differ. The high capacity of Escherichia coli to form populations resistant to anti-microbial medications, including pan-resistant ones, also is a serious problem for science and public health. The object of study was a collection of isolates of E. coli (n = 112), separated from 112 children aged under 5 years with clinical manifestations of acute intestinal infection, food toxic infection hemocolitis and diarrhea of obscure etiology in Yaroslavl in 2015-2017. Initially, the strains of E. coli were tested using diagnostic agglutinating coli-serums and then using reagents' kit «AmpliSens®Escherichiosis-FL» for detection and differentiation DNAof diarrheagenic bacteria coli and also with specific oligonucleotide primers to genes of virulence and O-serum group belonging. The obtained data permitted to determine belonging of analyzed strains of E. coli to four sub-groups: ЕНЕС (n = 9), EPEC (n = 17), ETEC (n = 1) и EAgEС (n = 1). All of them were agents of genes of pathogenicity specific for every pathogroup. The most numerous group EPEC was represented by strains of five serogroups with dominating among them serogroup O26 (9 strains). Therefore, studying collection of strains of diarrheagenic Escherichia isolated during 2015-1017 in Yaroslavl from children aged under 5 years with acute intestinal infections permitted to demonstrate efficiency of application of molecular genetic methods of analysis for characterizing E. coli i.e. establishment their serogroups, detection of genes of virulence and attributing to pathogroups.

Promising ichthyologic studies in Lake Baikal: fundamental and applied aspects

Promising areas of ichthyologic research in Lake Baikal have been examined. Particular attention is being paid to the technology of remote fsh census, including hydroacoustic methods and the method of quantitative environmental DNA analysis. These technologies may provide information on the status of artifcially reproducible populations in reservoirs for developing a proper fshing strategy. The areas of application of molecular genetic methods for solving applied problems in creating the biological bases of fsheries, studying diseases, associated microflora and parasitic fauna, as well as for identifying fsh feed objects have been determined. Studies aimed at restoring and maintaining the populations of rare and endangered fsh species using artifcial reproduction methods, including breeding, hybridization, hormonal stimulation of spawning producers, cryopreservation of sexual products, individual tagging, etc., are discussed. Prospects of mobile remote underwater video monitoring systems, the EthoStudio software package and the holography method to simulate sensory reaction in studying mechanisms of adaptations of fsh in natural environment and to develop criteria for assessing their stability in aquaculture conditionshave been reviewed. Combining ethological studies and in-depth integrated morphological, molecular-genetic and physiological-biochemical screening of Baikal fsh that would allow to better understand fundamental evolutionary processes responsible for the formation of behavioral adaptations, creation and maintenance of genetic diversity within and between populations in the natural environment, as well as developing recommendations on the introduction of state-of-theart scientifc monitoring approaches into aquaculture are very promising.

Application of molecular genetic methods for species Equus caballus – Literature review

In this study our aim was to provide a comprihensive overview of the most commonly used methods in molecular genetic studies related to Equus caballus. Thus we are dealing with the D-loop region of mitochondrial DNA, with microsatellites and also with single nucleotid polimorphism as SNP. The advantages and drawbacks of each method were also explored.

Application of molecular genetic methods for birds (literature review)

Ornithology studies have been extended by molecular genetic techniques. In this paper we are dealing with the most common use of areas. Our basic aim is to give a comprehensive view of the most commonly used methods in ornithological studies, including the available results by their use. We also deal with the following areas: an essential step during examination namely the applicability of sample collecting methods, and the unique identification, also the sex determination, methodology of DNA barcoding, as well as the role of molecular methods to protect endangered species. We discussed the advantages and disadvantages of the methods, such as the current trend for each method.

Historical overview of methods for sex determination in birds

Determining the sex in birds is very difficult, primarily because over 50% of species is monomorphic (no morphological differences between the sexes). Before the application of molecular genetic methods, there were used numerous methods all of which were un?reliable. Because of the importance of the analyses, they have to be reliable, economical, safe and prompt. Highly conserved CHD gene is defined in 1995. on W chromosome in birds, while on Z chromosome it was defined two years later. The difference in the length of the intronic sequences of CHD gene of Z and W chromosomes enables the distinguishing of the sexes after amplification of specific fragments by the application of specific primers. Molecular genetic methods have the supremacy over all the other methods because, ex?cept for being safe (both for birds and people), they provide reliable results, and also can be applied in all bird species.

Molecular detection and investigation of the presence of dirofilariosis causative agent in dogs on the territory of Belgrade

Dirofilariosis in dogs is a parasitic disease caused by the strains Dirofilaria immitis and D. Repens. Dirofilaria immitis parasitizes in the right side of the heart and pulmonary artery, causing considerable functional impairment of the heart, while adult forms of D. repens mostly parasitize in subcutaneous tissue causing minor skin damage. In this investigation, during the period from May to November 2015, 60 dogs of different race from the territory of Belgrade were examined. Ater the clinical examination, the blood was sampled for microfilariae detection by Knott test, as well as for molecular detection of the agent. Main clinical signs were: fatigue during exersise, shortness of breath and dry cough, anorexia, rapid heartbeat and skin changes. Microfilariae were detected in 22 dogs (37%) by modified Knott test. By molecular genetic method there was determined the prevalence of D. immitis 42%, and D. repens 22%. In this investigation 7 dogs were negative on Knott test, but by PCR method they showed a positive reaction on the presence of species of the genus Dirofilaria. On the basis of the obtained results, the application of molecular-genetic methods is justified in accurate and reliable species identification of Dirofilaria genus.

Development and Utilization of Plant Genetic Resources for Plant Breeding

For at least the last 10,000 years humans have been selecting plants that performed better for them across a wide range of criteria including fruit size, number of fruit per plant, adaptation to specific environments, resistance to certain pests, and many other characteristics. As the demand for agricultural productivity continues to rise, there is an increasing pressure on plant breeders to develop new varieties that are higher yielding and able to produce in more challenging environments. In recent years, there has been a significant increase in the application of molecular genetic methods for assessing the conservation and use of plant genetic resources that provides the new ways for plant breeding, for example, the high-throughput sequencing technology provides the possibility of analysing high numbers of samples over smaller periods of time. Currently, molecular breeding is standard practice in many crops, and a diverse collection of plant genetic resources and these resources remain accessible to breeders is important.This article describes the exploitation of plant genetic resources and modern plant breeding, introduces the application of molecular techniques in plant breeding and perspectives the significant of development and utilization of plant genetic resources for plant breeding.

Molecular genetic evidence for alternative reproductive strategies in North American pitvipers (Serpentes: Viperidae): long-term sperm storage and facultative parthenogenesis

The first documentation of facultative parthenogenesis (FP) in non-avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live-bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon contortrix), and the first genetically confirmed case of long-term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond-backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 934–942.

Assessing Plant Genetic Diversity by Molecular Tools

This paper is an overview of the diverse, predominantly molecular techniques, used in assessing plant genetic diversity. In recent years, there has been a significant increase in the application of molecular genetic methods for assessing the conservation and use of plant genetic resources. Molecular techniques have been applied in the analysis of specific genes, as well as to increase understanding of gene action, generate genetic maps and assist in the development of gene transfer technologies. Molecular techniques have also had critical roles in studies of phylogeny and species evolution, and have been applied to increase our understanding of the distribution and extent of genetic variation within and between species. These techniques are well established and their advantages as well as limitations have been realized and described in this work. Recently, a new class of advanced techniques has emerged, primarily derived from a combination of earlier, more basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques, in order to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. Some of the advanced marker techniques utilize newer classes of DNA elements, such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The most important and recent advances made in molecular marker techniques are discussed in this review, along with their applications, advantages and limitations applied to plant sciences.

Morphological likeness of the skeletal remains in a Central European family from 17th to 19th century

In spite of a recent preferential application of molecular genetic methods to kinship determination of anonymous human skeletal remains, the classical anthropological methods cannot be rejected as they are simple, quick and give access to a large part of a genome. This paper deals with the extent of morphological skeletal similarity in persons of known genealogical relationship. The skeletal remains of eight individuals from the family tomb of the Swéerts-Sporck's noble family in castle Kuks, East Bohemia, Czech Republic were analysed. Basic personal details, as well as data on their genealogical relationship, were available. Individuals were compared according to 173 anatomical variants – epigenetic traits, 90 of which were located on the skull and 83 on the postcranial skeleton. For each trait the percentile coincidence and/or difference were calculated. We observed the highest coincidence between the father and his son. These two individuals showed both closest correlation in the presence and the least difference in the occurrence of anatomical variants, as well as a high value of paternal probability. Clear kinship was also detected among cousins of the same or opposite sex. However, kinship between brother and sister was not so evident. The greatest difference was observed amongst biologically unrelated family members such as women who married into the family. The individuals under investigation showed a significantly higher occurrence of three among four traits of the sella turcica ( ponticulus carotico-clinoideus, ponticulus interclinoideus, taenia interclinoidea; 99% confidence). A significantly higher occurrence of the ponticuli basales ossis sphenoidalis, palatine torus and the costal articular surface on the body of 7th cervical vertebrae was also found (95% confidence). Our results, therefore, suggest that these morphological variants might be considered as family-specific traits.

HIV/AIDS epidemic features and trends in Saudi Arabia

BACKGROUNDMore than 60 million persons have been infected since AIDS was described in 1981. By the end of 2003, an estimated 40 million individuals were living with HIV globally. The surveillance of HIV/AIDS still faces challenges in Saudi Arabia. This study describes the pattern and characteristics of HIV/AIDS cases in Saudi Arabia.METHODSThis descriptive analytic study describes the reported HIV/AIDS data for all notifiable cases during the period 1984 to 2003 in the Kingdom of Saudi Arabia. Case definitions based on ELISA and Western Blot tests were used. Age, sex and regional distribution were studied in addition to survival rates.RESULTSAs of 2003, 1743 Saudi nationals and 6064 non-Saudi HIV cases were reported. Among Saudis, 872 were AIDS cases. Males accounted for 1329 HIV infections, comprising 77%, with a male-to-female ratio of about 3:1. Adults 15–49 years constituted 78% of cases, including 46% of cases infected through sexual activity, while 33% of reported HIV seropositive cases had already died. Most cases (67%) were registered in Jeddah, Riyadh and Dammam. Infection through blood transfusion declined with no reported cases since 2001.CONCLUSIONThe number of reported HIV infections in KSA has increased in the last few years. Using the coverage rate estimating method, the number of Saudi HIV infections since the emergence of the epidemic was estimated to be 2640 cases as of the beginning of the year 2004. The estimated number of HIV infections is almost one and one-half times the reported number, indicating that a problem of underreporting of HIV/AIDS cases still exists.

Application of molecular genetic methods in diagnostics and epidemiology of food‐borne bacterial pathogens

Salmonella enterica, Campylobacter and Yersinia species, Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Clostridium perfringens are the bacterial pathogens constituting the greatest burden of food-borne disease in Finland. Several molecular genetic methods have been applied to diagnose, discriminate and survey these bacteria. PCR, PCR-RFLP and PFGE are the most widely and successfully used. However, these methods are unable to replace conventional and internationally standardised phenotyping. Electronic database libraries of the different genomic profiles will enable continuous surveillance of infections and detection of possible infection clusters at an early stage. Furthermore, whole-genome sequence data have opened up new insights into epidemiological surveillance. Laboratory-based surveillance performed in a timely manner and exploiting adequate methods, and co-operation at local, national and international levels are among the key elements in preventing food-borne diseases. This paper reviews different applications of molecular genetic methods for investigating enteric bacterial pathogens and gives examples of the methods successfully used in diagnostics and epidemiological studies in Finland.

Application of molecular genetic methods in macrolide, lincosamide and streptogramin resistance diagnostics and in detection of drug‐resistant Mycobacterium tuberculosis

Antimicrobial susceptibility testing has traditionally been based on measurements of minimal inhibitory concentrations of antimicrobials. Molecular genetic studies on antimicrobial resistance have produced a great deal of genetic information which can be used for diagnosis of antimicrobial resistance determinants. Bacteria can acquire resistance to macrolides, lincosamides and streptogramin antibiotics by modification of the target site of the drugs, by active efflux of the drugs, and by inactivation of the drugs. The genetic backgrounds of these resistance mechanisms are well known and several molecular methods for detection of resistance determinants have been developed. Outbreaks of multidrug-resistant tuberculosis have focused international attention on the emergence of Mycobacterium tuberculosis strains that are resistant to antimycobacterial agents. Knowledge of the antimycobacterial resistance genetics and progress in molecular methods has made it possible to develop rapid molecular methods for susceptibility testing. This review presents the genetic background of drug resistance and introduces some methods for genotypic susceptibility testing.