D. Prosser, I. Raja, Kelly Kolkiewicz, A. Milano, D. Love
{"title":"Clinical Validation of a Whole Exome Sequencing Pipeline","authors":"D. Prosser, I. Raja, Kelly Kolkiewicz, A. Milano, D. Love","doi":"10.5772/intechopen.93251","DOIUrl":"https://doi.org/10.5772/intechopen.93251","url":null,"abstract":"Establishing whole exome sequencing (WES) in an accredited clinical diagnostic space is challenging. The validation (as opposed to verification) of an approach that will lead to clinical reports requires adhering to international guidelines and recommendations and developing a robust analytical pipeline that can scale due to the increasing clinical demand for comprehensive gene screening. This chapter will present a step-wise approach to WES validation that any laboratory can follow. The focus will be on highlighting the pivotal technical issues that must be addressed in validating WES and the analytical tools and QC metrics that must be considered before implementing WES in a clinical environment.","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"35 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80476633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Integrating Evolutionary Genetics to Medical Genomics: Evolutionary Approaches to Investigate Disease-Causing Variants","authors":"U. Sezerman, T. Bozkurt, F. S. Isleyen","doi":"10.5772/intechopen.92738","DOIUrl":"https://doi.org/10.5772/intechopen.92738","url":null,"abstract":"In recent years, next-generation sequencing (NGS) platforms that facilitate generation of a vast amount of genomic variation data have become widely used for diagnostic purposes in medicine. However, identifying the potential effects of the variations and their association with a particular disease phenotype is the main challenge in this field. Several strategies are used to discover the causative mutations among hundreds of variants of uncertain significance. Incorporating information from healthy population databases, other organisms’ databases, and computational prediction tools are evolution-based strategies that give valuable insight to interpret the variant pathogenicity. In this chapter, we first provide an overview of NGS analysis workflow. Then, we review how evolutionary principles can be integrated into the prioritization schemes of analyzed variants. Finally, we present an example of a real-life case where the use of evolutionary genetics information facilitated the discovery of disease-causing variants in medical genomics.","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"234 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76803948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"New Perspectives in Personalization of Therapy for Hematological Cancers","authors":"M. Rogalińska","doi":"10.5772/intechopen.91957","DOIUrl":"https://doi.org/10.5772/intechopen.91957","url":null,"abstract":"A progress in treatment of hematological cancers was achieved. Unfortunately, some youngsters, because of rare genetic alterations that are not easy to detect, as well as heavily pretreated old patients, because of coexisting diseases that lead to changes in patient metabolism, do not respond to therapy. Moreover, sometimes familiar diversities and alterations on genetic or epigenetic level that could be transferred on diversities in metabolism or cell signaling might be a reason why patients do not respond to therapy. Interestingly, for older patients a resistance to therapy could also occur as a reason of drug cross-reactivity. For designing of effective anticancer therapy for patient with chronic lymphocytic leukemia before drug administration, patient’s leukemic cell response to anticancer drug(s) should be checked. Moreover, for patient response to treatment, also drugs prescribed previously by other medical doctors or even patients’ diet could be important for achieving therapeutic success of therapy. Therefore it is important to choose the effective drugs before their administration to patient that will improve treatment efficacy and exclude resistance to therapy. It must be stated that the special attention for personalized therapy tests should be focused on patients previously resistant to therapy, more sensitive to drugs or heavily pretreated.","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85875796","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Molecular Medicines for Parasitic Diseases","authors":"Bhawana Singh","doi":"10.5772/intechopen.91956","DOIUrl":"https://doi.org/10.5772/intechopen.91956","url":null,"abstract":"Being the cause for significant amount of morbidities and mortalities, parasitic diseases remain the major challenge for the healthcare community due to the limitations associated with the current chemotherapeutics. Drug discovery/invention can be achieved by collaborative efforts of biotechnologists and pharmacists for identifying potential candidates and successfully turn them into medicine for improving the healthcare system. Although molecular medicine for disease intervention is still in its infancy, however, significant research works and successful trials in short span of time have made it broadly accepted among the scientific community. This chapter identifies different molecular medicine approaches for dealing with parasites that have been coming up on the horizon with the new technological advances in bioinformatics and in the field of omics. With the better understanding of the genomics, molecular medicine field has not only raised hopes to deal with parasitic infections but also accelerated the development of personalized medicine. This will provide a targeted approach for identifying the druggable targets and their pathophysiological importance for disease intervention.","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"5 1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82834761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Landscape Genetics: From Classic Molecular Markers to Genomics","authors":"E. R. Konzen, M. Zucchi","doi":"10.5772/intechopen.92022","DOIUrl":"https://doi.org/10.5772/intechopen.92022","url":null,"abstract":"Landscape genetics combines population genetics and landscape ecology to understand processes that shape the distribution and organization of human, animal, or plant populations. This field of genetics emerged from the availability of several studies with classical molecular markers, such as isozymes, RAPD, AFLP, and microsatellites. Population genetic studies enabled the detection of population structure with those markers, but a more comprehensive analysis of natural populations was only possible with the development of statistical methods that combined both molecular data and environmental variables. Ultimately, the rapid development of sequencing technologies allowed studies at the genomic level, augmenting the resolution of association with environment factors. This chapter outlines basic concepts in landscape genetics, the main statistical methods used so far, and the perspectives of this field of knowledge into strategies for conservation of natural populations of plant and animal species. Moreover, we briefly describe the application of the field to understand historical human migration processes as well as how some diseases are spread throughout the world.","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2020-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75478474","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"DNA-mediated gene transfer.","authors":"A. J. O'Donnell","doi":"10.1002/9783527678679.dg03327","DOIUrl":"https://doi.org/10.1002/9783527678679.dg03327","url":null,"abstract":"","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"16 1","pages":"259-66"},"PeriodicalIF":0.0,"publicationDate":"2020-02-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83184701","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Assay of air sample eluates.","authors":"Anne Renström, Susan Gordon","doi":"10.1007/978-1-59745-366-0_18","DOIUrl":"https://doi.org/10.1007/978-1-59745-366-0_18","url":null,"abstract":"<p><p>After air sampling and elution, the air sample eluate contains an unknown amount of allergens together with other materials. The proteins of interest can be quantified using immunoassays, which are sensitive, economical, and can be used for high throughput. However, the amount of antigen or allergen in an air sample may be very low and consequently the assays must be very sensitive and specific. Immunoassays use antibodies both to capture and visualize the chosen antigen. High specificity and sensitivity can best be achieved by the use of purified, characterized, and specific antibodies. It is possible to choose between a wide variety of assay setups and reagents. The method described here has been developed for the measurement of airborne rodent allergens. It is a noncompetitive, two-site (sandwich) EIA that utilizes polyclonal antibodies. The detection system uses biotin and streptavidin for increased sensitivity and horseradish peroxidase as the substrate with 3,3',5,5'-tetramethylbenzidine (TMB) for rapid color development and high sensitivity.</p>","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"138 ","pages":"217-25"},"PeriodicalIF":0.0,"publicationDate":"2008-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/978-1-59745-366-0_18","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27523989","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Conjugation of haptens.","authors":"Ranulfo Lemus, Meryl H Karol","doi":"10.1007/978-1-59745-366-0_14","DOIUrl":"https://doi.org/10.1007/978-1-59745-366-0_14","url":null,"abstract":"<p><p>Many naturally occurring proteins, peptides, carbohydrates, nucleic acids, and lipids, as well as synthetic peptides, are successful immunogens. To elicit an immune response, a compound must contain an antigenic determinant or epitope and must be of sufficient size to initiate lymphocyte activation necessary for an antibody response. In practice, small chemical compounds (haptens) are generally not good immunogens. However, when attached to macromolecules (carriers), they can become immunogenic. An immunogen must have epitopes that can be recognized by antigen-presenting cells and a T-cell receptor, and it must be degradable. Haptens and corresponding hapten-carrier conjugates have been essential to the development of sensitive quantitative and qualitative immunoassays. In the design of hapten conjugates, consideration must be given to the hapten, the carrier, the coupling strategy, and the hapten density because the amount of hapten attached to the carrier influences the strength of the immune response directed toward the newly created antigenic determinant. Hence the haptenic density of the conjugate is also important in the development of immunoassays. The optimal epitope density of a conjugate to elicit either a strong immune response or provide the best immunoassay is dependent on the structure of the epitope and the nature of the immunoassay. The aim of this chapter is to describe the diverse techniques used to couple haptens to carriers and provide guidance in the selection of the most appropriate procedure for a particular hapten.</p>","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"138 ","pages":"167-82"},"PeriodicalIF":0.0,"publicationDate":"2008-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/978-1-59745-366-0_14","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27522385","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Understanding of the molecular mechanisms of allergy.","authors":"Meinir Jones","doi":"10.1007/978-1-59745-366-0_1","DOIUrl":"https://doi.org/10.1007/978-1-59745-366-0_1","url":null,"abstract":"<p><p>The prevalence of allergic disease has dramatically increased over the past 30 years in Westernised countries. It is unlikely that the rapid increase in the prevalence of allergic disease is the result of genetic changes, which highlights the importance of environmental factors in the development of allergic disease. The 'hygiene hypothesis' was put forward in 1989 and focused attention on the notion that exposure to microbes and their products in early life can modify the risk for development of allergic disease. Infections were thought to polarize the immunological response towards a Th2-mediated immune responses causing allergic disease. However it is likely that the Th1/Th2 imbalance is too simplistic to explain the increased prevalence of allergic disease. Current research is focusing on understanding the role of T regulatory cells in inducing a state of tolerance and the resulting modified Th2 response observed in natural and induced tolerance.</p>","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"138 ","pages":"1-15"},"PeriodicalIF":0.0,"publicationDate":"2008-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/978-1-59745-366-0_1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27522435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A method to assay inhibitors of DNA polymerase IIIC activity.","authors":"Michelle M Butler, George E Wright","doi":"10.1007/978-1-59745-246-5_3","DOIUrl":"https://doi.org/10.1007/978-1-59745-246-5_3","url":null,"abstract":"<p><p>The need for new drugs to treat infections caused by antibiotic-resistant bacterial strains has prompted many studies to identify novel targets in pathogenic bacteria. Among the three DNA polymerases expressed by bacteria, one of these, designated pol III, is responsible for DNA replication and growth of bacteria and, therefore, warrants consideration as a drug target. However, the pol III enzymes of Gram-positive and Gram-negative species are quite different, and the Gram-positive enzyme pol IIIC has been more extensively studied as a drug target than the Gram-negative enzyme pol IIIE.DNA polymerases are unique enzymes with respect to the five substrates (four dNTPs, one of which is radiolabeled, and primer:template DNA) that they typically utilize. Variations of the assay, e.g., by leaving out one dNTP but allowing measurable incorporation of the remaining substrates, or use of homopolymer primer:templates, may be used to simplify the assay or to obtain mechanistic information about inhibitors. Use of gel analysis of primer extension assays can also be applied to study alternate substrates of DNA polymerases. Methods to isolate pol IIIC from Gram-positive bacterial cells and to clone and express the polC gene are described in this chapter. In addition, the assay conditions commonly used to identify and study the mechanism of inhibitors of pol IIIC are emphasized.</p>","PeriodicalId":18460,"journal":{"name":"Methods in molecular medicine","volume":"142 ","pages":"25-36"},"PeriodicalIF":0.0,"publicationDate":"2008-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1007/978-1-59745-246-5_3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"27404312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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