Application Of Molecular Genetics Research Articles

The role of molecular genetics in livestock production

Genetic variations that lead to easy-to-identify phenotypic changes have always been of interest to livestock breeders since domestication. Molecular genetics has opened up possibilities for identifying these variations and understanding their biological and population effects. Moreover, molecular genetics is part of the most diverse approaches and applications in animal production nowadays, including paternity testing, selection based on genetic variants, diagnostic of genetic diseases, reproductive biotechniques, fraud identification, differentiation of hybrids, parasite identification, genetic evaluation, diversity studies, and genome editing, among others. Therefore, the objective of this review was to describe the different applications of molecular genetics in livestock production, contextualising them with examples and highlighting the importance of the study of these topics and their applications.

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Endonuclease Cas9–Mediated Homology-Independent Integration for Generating Quality Control Materials for Clinical Molecular Genetic Testing

Genome-edited human cell lines are important resources for producing quality control materials for clinical molecular genetic testing. Generating cell lines with defined mutations through homology-directed repair-based methods are inefficient and can lead to unwanted insertions and deletions in the target loci. Nonhomologous end joining in the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) system was harnessed to generate genome-engineered cell lines harboring target mutations. Donor plasmids containing target sites for the single guide RNA (sgRNA) and homologous DNA fragments harboring important cancer gene mutations were cotransfected with the Cas9/sgRNA vector into wild-type human cells. The introduced mutations were validated in-house and in 44 laboratories using various techniques, including next-generation sequencing. Exogenous sequences containing the target mutations were efficiently integrated into the ALK receptor tyrosine kinase (ALK) locus in HEK293T and A549 cells. Successful introduction of artificial mutations was confirmed via both Sanger sequencing and the amplification refractory mutation system. Results of external pilot testing revealed that the DNA samples derived from genome-edited cell lines were widely applicable across multiple platforms and laboratories. This study demonstrates that CRISPR/Cas9-induced nonhomologous end joining is a valuable and novel method for generating artificial mutants for use in quality control applications in clinical molecular genetics.

Electronic Properties of Synthetic Shrimp Pathogens-derived DNA Schottky Diodes

The exciting discovery of the semiconducting-like properties of deoxyribonucleic acid (DNA) and its potential applications in molecular genetics and diagnostics in recent times has resulted in a paradigm shift in biophysics research. Recent studies in our laboratory provide a platform towards detecting charge transfer mechanism and understanding the electronic properties of DNA based on the sequence-specific electronic response, which can be applied as an alternative to identify or detect DNA. In this study, we demonstrate a novel method for identification of DNA from different shrimp viruses and bacteria using electronic properties of DNA obtained from both negative and positive bias regions in current-voltage (I–V) profiles. Characteristic electronic properties were calculated and used for quantification and further understanding in the identification process. Aquaculture in shrimp industry is a fast-growing food sector throughout the world. However, shrimp culture in many Asian countries faced a huge economic loss due to disease outbreaks. Scientists have been using specific established methods for detecting shrimp infection, but those methods do have their significant drawbacks due to many inherent factors. As such, we believe that this simple, rapid, sensitive and cost-effective tool can be used for detection and identification of DNA from different shrimp viruses and bacteria.

Sodium channels.

In 2000, with the completion of the human genome project, nine related channels were found to comprise the complete voltage-gated sodium gene family and they were renamed NaV1.1–NaV1.9. This millennial event reflected the extraordinary impact of molecular genetics on our understanding of electrical signalling in the nervous system. In this review, studies of animal electricity from the time of Galvani to the present day are described. The seminal experiments and models of Hodgkin and Huxley coupled with the discovery of the structure of DNA, the genetic code and the application of molecular genetics have resulted in an appreciation of the extraordinary diversity of sodium channels and their surprisingly broad repertoire of functions. In the present era, unsuspected roles for sodium channels in a huge range of pathologies have become apparent.

Bridging molecular genetics and participatory research: how access and benefit‐sharing stimulate interdisciplinary research for tropical biology and conservation

AbstractMolecular genetics research can benefit efforts to conserve the genetic diversity of tropical plant species. Clear and efficient procedures are needed to access DNA samples, while respecting tropical countries’ and local communities’ rights on genetic resource usage. The Nagoya Protocol on Access and Benefit‐Sharing, which took effect in 2014, provides an opportunity to establish such procedures. However, scientists are concerned that its emphasis on monetary gains restricts research focused on scientific, societal, and environmental benefits. Despite much political and scientific debate, few concrete cases have demonstrated the practical functioning of the Nagoya Protocol. This paper describes the first application of the Protocol in Guatemala, where it was used to grant permission to a non‐commercial study on gene flow in mahogany (Swietenia macrophylla King) populations in the Maya Biosphere Reserve of Petén. On the basis of this study, we discuss five strategies to enhance the application of molecular genetics to conservation biology under the Nagoya Protocol: (1) generate short and standardized procedures; (2) enable science communication; (3) cultivate a common understanding between users, providers, and potential beneficiaries; (4) involve local research and practitioner organizations; and (5) integrate participatory research. Positive societal views on the application of molecular genetics to conservation biology generate further support for work in this discipline and promote adoption of research results for the conservation of genetic diversity of tropical plant species.

Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications

BackgroundSeveral high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications.ResultsWe developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection.ConclusionTo facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.

In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris) Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS) Genes.

Mature oak (Quercus spp.) leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS) from pin oak (Quercus palustris), whose full genome has not yet been sequenced.

Molecular diagnosis of pediatric tumors

<p>Pediatric tumors are challenging in the context of best diagnosis, treatment, and prognosis. For tumors which have a genetic association or a cancer predisposition syndrome, the prognosis depends on accurate diagnosis. The application of molecular genetics to pediatric tumors has resulted in better diagnostic and prognostic factors for patient management. Molecular diagnostic techniques, such as reverse transcription polymerase chain reaction and fluorescence in situ hybridization (FISH), have become important tests for childhood tumors. Targeted therapies are aimed at specific translocations which are detected by FISH. Molecular techniques help in monitoring of minimal residual disease in childhood tumors.</p>

Some pioneers of European human genetics.

Some of the pioneers of human genetics across Europe are described, based on a series of 100 recorded interviews made by the author. These interviews, and the memories of earlier workers in the field recalled by interviewees, provide a vivid picture, albeit incomplete, of the early years of human and medical genetics. From small beginnings in the immediate post-World War 2 years, human genetics grew rapidly across many European countries, a powerful factor being the development of human cytogenetics, stimulated by concerns over the risks of radiation exposure. Medical applications soon followed, with the recognition of human chromosome abnormalities, the need for genetic counselling, the possibility of prenatal diagnosis and later, the applications of human molecular genetics. The evolution of the field has been strongly influenced by the characters and interests of the relatively small number of founding workers in different European countries, as well as by wider social, medical and scientific factors in the individual countries.European Journal of Human Genetics advance online publication, 10 May 2017; doi:10.1038/ejhg.2017.47.

Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines.

Small-molecule fluorophores, such as fluorescein and rhodamine derivatives, are critical tools in modern biochemical and biological research. The field of chemical dyes is old; colored molecules were first discovered in the 1800s, and the fluorescein and rhodamine scaffolds have been known for over a century. Nevertheless, there has been a renaissance in using these dyes to create tools for biochemistry and biology. The application of modern chemistry, biochemistry, molecular genetics, and optical physics to these old structures enables and drives the development of novel, sophisticated fluorescent dyes. This critical review focuses on an important example of chemical biology-the melding of old and new chemical knowledge-leading to useful molecules for advanced biochemical and biological experiments.

Recent Updates on Molecular Genetic Engineering Approaches and Applications of Human Therapeutic Proteins

Therapeutic proteins are engineered proteins produced in the laboratory for pharmaceutical use. With the advent of recombinant DNA technology, the proteins can be generated in specific host cells under defined conditions. In the process of production of genetically engineered animals, the gene of interest can be added at a single cell stage to produce a cloned animal from genetically engineered cells. Several recombinant cytokines, clotting factors etc have been licensed and are currently being utilized for the treatment of cancer, infectious diseases, hemophilia, anemia, multiple sclerosis, and hepatitis B/C. Therapeutic proteins that are useful for human are successfully produced in poultry as well as in livestock animals. However, the fastest growing class of therapeutic proteins are antibodies especially monoclonal antibodies (mAb), the most important class of therapeutic protein with the potential to generate significant revolution in terms of clinical success rate. Here, we review the most recent clinical advances in the field of emerging and existing therapeutic proteins.

Establishing a high throughput screening method for large scale phenotyping of castor genotypes for resistance to Fusarium wilt disease

Castor (Ricinus communis L.) is an important oilseed crop providing raw materials for many industries. Its cultivation is seriously affected by the wilt disease caused by Fusarium oxysporum f. sp. ricini. In India, no cultivar could be released for cultivation without wilt resistance. Breeding for wilt resistance is hampered due to the limitation on number of genotypes that can be screened in a traditional ‘sick plot (field) method’. In the pursuit of establishing a high throughput screening method, we evaluated four different methods of artificial inoculation namely seed soaking, soil drenching, root dip and sick pot in glasshouse condition for their efficiency using a panel of eight genotypes with known disease reaction. The results showed that ‘sick pot method’ was the most ideal for accurate identification of resistance or susceptibility in plants in a short time with relative ease. In order to further validate the results, a large set of 132 castor inbred lines were evaluated in sick pot. Based on days-to-death data of the inbred lines, a scoring system was developed to suitably characterize the degree of resistance. Screening of the same set of inbred lines in the sick field produced similar results but moderate and highly resistant genotypes could not be differentiated suggesting the advantage of sick pot method over field screening. The screening method established and the set of resistant or susceptible inbred lines identified in this study could be of immense use in basic research concerning host-pathogen interactions, molecular genetics and breeding applications in castor.

Latest developments in precancerous lesions of hepatocellular carcinoma.

Hepatocarcinogenesis in human chronic liver diseases is a multi-step process in which hepatic precancerous lesions progress into early hepatocellular carcinoma (HCC) and progressed HCC, and the close surveillance and treatment of these lesions will help improve the survival rates of patients with HCC. The rapid development and extensive application of imaging technology have facilitated the discovery of nodular lesions of ambiguous significance, such as dysplastic nodules. Further investigations showed that these nodules may be hepatic precancerous lesions, and they often appear in patients with liver cirrhosis. Although the morphology of these nodules is not sufficient to support a diagnosis of malignant tumor, these nodules are closely correlated with the occurrence of HCC, as indicated by long-term follow-up studies. In recent years, the rapid development and wide application of pathology, molecular genetics and imaging technology have elucidated the characteristics of precancerous lesions. Based on our extensive review of the relevant literature, this article focuses on evidence indicating that high-grade dysplastic nodules are more likely to transform into HCC than low-grade dysplastic nodules based on clinical, pathological, molecular genetic and radiological assessments. In addition, evidence supporting the precancerous nature of large cell change in hepatitis B virus-related HCC is discussed.

Applications of Molecular Genetics to the Study of Asthma.

Asthma is a multifactorial disease. This fact, associated to the diversity of asthma phenotypes, has made difficult to obtain a clear pattern of inheritance. With the huge development of molecular genetics technologies, candidate gene studies are giving way to different types of studies from the genomic point of view.These approaches are allowing the identification of several genes associated with asthma. However, in these studies, there are some conflicting results between different populations and there is still a lack of knowledge about the actual influence of the gene variants. Some confounding factors are, among others, the inappropriate sample size, population stratification, differences in the classification of the phenotypes, or inadequate coverage of the genes.To confirm the real effect of the reported associations, it is necessary to consider both the genetic and environmental factors and perform functional studies that explain the molecular mechanisms mediating between the emergence of gene variants and the development of the disease.The development of experimental techniques opens a new horizon that allows the identification of major genetic factors of susceptibility to asthma. The resulting classification of the population groups based on their genetic characteristics, will allow the application of specific and highly efficient treatments.

Revision of the genus Hydroides (Annelida: Serpulidae) from Australia.

Hydroides Gunnerus, 1768 is the largest and one of the economically most important genera of calcareous tubeworms (Serpulidae, Annelida) that includes a number of notorious fouling and bioinvading species. Although the representatives of the genus are typically found in shallow waters of tropical and subtropical areas worldwide, the species composition of the genus in Australia has never been revised. We conducted the first detailed regional taxonomic revision of Hydroides species based both on the historical collections from Australian museums (Australian Museum, Museum Victoria, South Australian Museum, Western Australian Museum, Queensland Museum, and Museum and Art Gallery of Northern Territory) and newly collected material from New South Wales, Victoria, Queensland, Northern Territory, and Western Australia. In total, 25 species are currently considered valid in Australia, including three new species: H. amri n. sp. from NSW, SA, and Vic (previously referred to as H. cf. brachyacantha), as well as H. glasbyi n. sp. and H. qiui n. sp., both from NT, and two new records of H. furcifera and H. multispinosa for Australia. We have synonymised H. spiratubus with H. albiceps, and H. spiculitubus with H. tambalagamensis in this study. The status of the taxon H. cf. recta remains undecided. An identification key and diagnoses accompanied by original high-quality photographs for all species recorded in Australia are provided. Application of molecular genetics is needed to resolve the status of some problematic species.

Size-defined morphotypes in Zoanthus (Hexacorallia: Zoantharia) populations on shores in KwaZulu-Natal, South Africa.

Colonial zoanthids are a conspicuous feature of the subtropical rocky intertidal in KwaZulu-Natal but those of the genus Zoanthus have a confused taxonomy with 10, difficult to separate, nominal species described from the region. This paper presents an analysis of polyp size, measured as mean diameter determined photographically from the number of polyps occupying an area of 6 × 4 cm(2). The results, based on diameter frequency of 127 samples from five shores, indicate three populations (morphotypes) with means of 4.3 (SD ±0.53), 5.7 (SD ±0.70) and 8.4 (SD ±0.58) mm occurring in the approximate abundance ratios of 10:5:1, possibly corresponding to Zoanthus sansibaricus, Z. natalensis and Z. lawrencei. The underlying assumptions with regard to population structure (the number, size and degree of fragmentation of clones) and the normality of data are discussed, as are trans-oceanic larval dispersal, recruitment, and genetic connectivity. The essential, traditional species description in Zoanthus, using internal morphology, on its own may be an inadequate discriminator of species. The status of the few possibly valid species is discussed in relation to the present results and recent studies based on the application of molecular genetics. Thorough studies of the population structure (genets and ramets) and a taxonomic approach based on the combined use of morphology, genetic methods and statistically robust, quantitative morphometrics are proposed as a potential way forward.

Molecular immunohaematology round table discussions at the AABB Annual Meeting, Boston 2012.

Well trained, experienced serologists in transfusion medicine laboratories have been familiar with blood group serology for decades. With the advent of molecular immunohaematology, there is a need to adopt and embrace the clinical and diagnostic applications in order for patients to benefit from the advances that this technology offers1–3. We organised an international forum to discuss molecular immunohaematology concepts that may be challenging even for some established professionals in the field of serology. The objectives of the session were two-fold. First, the session allowed networking among immunohaematology professionals who have an interest in the application of molecular immuno-haematology and blood group genetics while meeting with leaders in the field of molecular immunohaematology. By giving input and asking questions, the participants could define their knowledge relative to the experience of the group as a whole. Second, the discussions and the input from experienced professionals were recorded. This approach allowed documentation of current knowledge, as well as acceptance and concerns of the participants. It is useful to gathering information in this field, because the perception at the level of the participants is critical for shaping the adoption of molecular immunohaematology. We provide a summary report of the items discussed and issues raised by the participants. The results describe the status of molecular immunohaematology within this large group of experienced professionals in transfusion medicine laboratories and can guide targeted educational efforts.

Bioinformatic approach in the identification of arabidopsis gene homologous in amaranthus

Bioinfomatics offers an efficient tool for molecular genetics applications and sequence homology search algorithms became an inevitable part for many different research strategies. Appropriate managing of known data that are stored in public available databases can be used in many ways in the research. Here, we report the identification of RmlC-like cupins superfamily protein DNA sequence than is known in Arabidopsis genome for the Amaranthus - plant specie where this sequence was still not sequenced. A BLAST based approach was used to identify the homologous sequences in the nucleotide database and to find suitable parts of the Arabidopsis sequence were primers can be designed. In total, 64 hits were found in nucleotide database for Arabidopsis RmlC-like cupins sequence. A query cover ranged from 10% up to the 100% among RmlC-like cupins nucleotides and its homologues that are actually stored in public nucleotide databases. The most conserved region was identified for matches that posses nucleotides in the range of 1506 up to the 1925 bp of RmlC-like cupins DNA sequence stored in the database. The in silico approach was subsequently used in PCR analysis where the specifity of designed primers was approved. A unique, 250 bp long fragment was obtained for Amaranthus cruentus and a hybride Amaranthus hypochondriacus x hybridus in our analysis. Bioinformatic based analysis of unknown parts of the plant genomes as showed in this study is a very good additional tool in PCR based analysis of plant variability. This approach is suitable in the case for plants, where concrete genomic data are still missing for the appropriate genes, as was demonstrated for Amaranthus.

Genomic Selection, a New Era for Pork Quality Improvement

Traditional breeding approaches apply sophisticated statistical tools such as best linear unbiased prediction (BLUP) to evaluate the genetic potential of animals for economically important traits using phenotype and pedigree information observed on the animal. However, the genetic gain achieved is relatively slow for traits with low-to-moderate heritability, or expensive to measure traits, such as those determined post-mortem e.g., pork quality. Nowadays, the availability of dense panels of DNA markers covering the whole genome along with powerful statistical tools has made genomic selection (GS) feasible in pigs. The large number of single nucleotide polymorphisms generated by high-throughput technologies can be used in GS to select superior animals with better meat quality. Many quantitative trait loci (QTL) affecting meat quality traits have been detected in pigs demonstrating the potential for this improvement. Genomic selection uses genome-wide markers so that all QTL are likely to be in linkage disequilibrium with at least one marker. Genomic selection sums the effects of markers covering the whole genome so that potentially all the genetic variance associated with the traits and explained by the markers are considered. This can greatly improve selection accuracy to accelerate genetic gain for pork quality traits. In this review, we discuss the genetic component underlying pork quality variation, statistical approaches for pork quality genomic prediction, and present recent highlights for their application in swine breeding programs. Firstly, we review how pork quality is integrated into breeding objectives. Secondly, we present approaches for application of molecular genetics in meat quality improvement. Additionally, we discuss the statistical methods for genomic prediction including ridge regression, Bayesian approaches, GBLUP, and combining genomic and traditional information using single-step BLUP. Finally, we review the strategies for their use in swine genetic improvement and management. In particular, we review the strategy for implementing GS in swine breeding programs to improve pork quality.

Comparative study of the influence of EDTA and sodium heparin on long term storage of cattle DNA.

Blood collection in heparin tubes for cytogenetic, and ethylenediaminetetraacetic acid (EDTA) tubes for molecular genetics applications respectively, are routine practices everywhere. If blood samples are required for cytogenetics as well as DNA work, two samples from each animal are usually collected, which leads to wastage of time and money. The present study tried to explore the possibilities of collecting a single blood sample in a heparinised tube for use in both applications. Two blood samples were collected from the same animals; one in a heparin tube and the other in an EDTA tube. DNA was extracted and stored at the same temperature and for the same durations. Comparative studies revealed that the DNA samples extracted from blood using these two different coagulants give more or less the same quality of results especially for polymerase chain reaction (PCR) based applications in cattle. The purpose of the present study was to establish the possibility of using heparin blood for chromosomal studies as well as for molecular biology. Such a practice will obviously save time and money in collecting samples in duplicate.