Summary for Molecular Biotechnology in Diagnostics: Step towards Precision Medicine
Current advancements in Molecular diagnostics via new techniques and instrumentation have led to various applications in diagnosing cancerous, neurological, genetic and microbial diseases. The ability to gather large amounts of information on human genes and study them have led to the field of genetics being at the forefront of medical research and practices in pharmacogenomics, chemo-informatics, and nutrigenomics. Through advanced amplification and screening techniques, scientists can now analyze numerous nucleic acid sequences at once contributing to the standardization of traditional biomarkers. Understanding the human genome and the relationship between genotype to phenotype have propelled the need for new molecular diagnostic screening techniques to detect abnormal mutations and disease-related genes. With the use of DNA microarray, proteomics, and single nucleotide polymorphisms analysis, scientists are able to develop a human genome map. This map, coupled with bioinformatics, has broadened the field of genetics and brought new applications to healthcare for managing a host of currently untreatable diseases and disorders.
Molecular Diagnostics have given researchers insight into how certain gene markers may predispose individuals to certain disease states. Also, clinicians are able to understand the role genes play in the mechanism of different diseases allowing for improved treatments. The diversity of gene mutations and vastness of gene-related disorders have made necessary the need for a variety of detection methods advancing the field of functional genomics. Therapies targeted at specific genes allow for improved and individual treatment plans specific to the patient. The possibility of using genotyping tools to screen individuals for their unique molecular signature will allow clinicians to predict the susceptibility of numerous diseases. Knowing which genetic variances drugs affect, clinicians can be more selective and specific in their drug choices. Targeted therapies have been used to treat certain cancers by recognizing certain cell expressions as specific targets for therapy. Using molecular techniques to analyze cancerous cells allow for researchers to discover abnormalities present in the cell that are absent in normal cells and develop targeted therapies directed at the specific abnormality. Molecular studies of diseases allow for early detection of disease states with a high specificity allowing for earlier medical intervention for the most optimal patient outcome.
The vast amount of data gathered on the human genome has allowed researchers to understand why some drugs work better on certain patients versus other patients with the same disease. The human genome sequence shows researchers specific variances among population and individuals and gives insight into the mechanism of drug variability among a population. Sequence differences in drug target proteins, drug metabolizing enzymes, and drug transporters can change drug efficacy, drug side effects, or both, to cause variable drug responses in individual patients. This new information has translated into other aspects of research including that of pharmacogenetics. Pharmaceutical companies and researchers can work to develop precise haplotyping techniques. This will give clinicians the ability to target specific treatments on an individual basis according to who will benefit from a certain type of drug versus another. This approach will allow for better management and a more cost-effective treatment approach. Nutritional genomics (nutrigenomics), which studies the effects of diet and genomics, is a new area of research helping clinicians understand the effects that ingested nutrients have on gene expression and gene regulation. This information can then be used to better understand how food is having beneficial or harmful health effects on an individual basis. Physicians can use this information to develop a personalized diet according to one’s diet-gene interaction needs. This new area of study will also begin to make the link between chronic diseases and diet.
Molecular Biotechnology is changing the way clinicians diagnose and screen for diseases, evaluate risk factors, and use drugs for specific targeted therapy. This allows for a new approach to medicine in which treatment is based on an individual’s unique genetic makeup. Future challenges include cost-effectiveness, accuracy, reproducibility, and choice of methodology. As gene-sequencing technology continues to develop, costs of instruments will begin to be justified, and the database of “disease-genes” will continue to grow and help clinicians better deliver targeted therapy to patients.
In the future, molecular diagnostics will always use PCR-based testing in clinical laboratories; however, researchers will not use PCR-based techniques to discover genome complexity. Researchers will be using integrated silicon chips mounted with biomolecules. This is an expensive technique to use. This technique though will save time and use fewer sample amounts than a PCR-based technique. The future will continue to bring in new technology and new techniques to the forefront in all laboratories around the world.
Summary for Molecular Genetic Testing and the Future of Clinical Genomics
Clinical molecular genetic testing is transforming personalized medicine, and it is appropriate for a range of applications; including rare disease diagnostics, predictive testing for common disorders, and pharmacogenetic testing. The circumstances of a specific genetic test used to depend on the age of the patient, family history, the nature of the disease or disorder and specimen availability. Many procedures used are either direct or indirect genetic analyses. Direct and indirect genetic analyses may use the same methodologies. Direct genetic testing looks at the presence or absence of a known genetic variant or variants that cause the disease. Indirect genetic testing compares DNA markers from an unaffected person to an affected person. Then the physician evaluates to see if the patient has the trait, but the trait does not cause the condition.
In clinical molecular diagnostics, targeted allele-specific mutation detection is also called target PCR, and it is a cheap and fast method to find a specific variant. However, targeted PCR has some disadvantages. It cannot detect any other valuable variants the disease might cause. An example of a target PCR is using a TaqMan assay to detect a variant. The TaqMan assay uses a hybridization probe that cleaves to the product, and the specific variant is detected using fluorescence. This procedure will still be in laboratories in the future because some facilities have limited resources or lack of advanced technology to do further in genetic testing.
Sanger sequencing has been the gold standard in genetics for many years in detecting small variants and point mutations which are small changes in the DNA sequence. Frederick Sanger developed a chain terminator method that is a modified version of the DNA replication process. It uses four fluorescence-labeled dideoxynucleotides; and they are ddATP, ddCTP, ddGTP, and ddTTP to stop the growing DNA chain because they lack the 3' OH group. The four labeled dideoxynucleotides are split up in four tubes, and then they are loaded for the capillary electrophoresis. The capillary electrophoresis sorts them by sizes which then fluoresce at a specific wavelength when they pass through a laser beam. Sanger sequencing does not have a fast turnaround time. However, Sanger sequencing has a high sensitivity and specificity at a low cost. This testing method alone is not adequate enough to diagnosis disorders.
There are three types of microarrays used in genotyping. They are array comparative genomic hybridization aka array CGH, phenotype-specific single-nucleotide polymorphisms (SNP) arrays, and genome-wide SNP arrays. Array CGH is used to detect structural and chromosomal changes such as deletions and duplications. It has average sensitivity and specificity. Phenotype-specific SNP array is used to determine if the patient has alleles that are known for certain phenotypes. It has a low cost. Genome-wide SNP array is used to see if the patient is at risk for multiple disorders and diseases. It will also help with pharmacogenetics tests. It has a low sensitivity and specificity. The cost is low, and it has a fast turnaround time. An example of a whole-genome wide SNP array is a 23andMe test.
Other tests used to detect structural and chromosomal variations are fluorescent in situ hybridization (FISH), southern blot and multiplex ligation-dependent probe amplification (MLPA) assays. FISH is a technique that uses a fluorescence microscope to find the fluorescent probes that bind to the complementary sequence of the chromosome. FISH has low sensitivity and specificity. It has a low cost and fast turnaround time. Southern blot is a method that detects specific DNA sequence and uses electrophoresis to separate fragments and is then transferred to a membrane. After the transfer, the specific fragment is detected by a hybridization probe. MLPA assays detect mosaic mutations and methylation status. Southern blot and MLPA assays both have high sensitivity and specificity. They have a low cost but a slow turnaround time.
Whole-exome (WES) and whole-genome (WGS) sequencing may become a first-line clinical test for some diagnostic cases because they look for variants in a range of rare disorders. WES looks for the targeted exons in the genome, and WGS looks at the whole genome. Genome sequencing may transform diagnostic approaches in large academic medical centers in which access to expensive and sophisticated tests are available but these resources do exist in all facilities. The greatest challenge to clinical genomics is the reliable interpretation of the multiple and novel variants found through genome sequencing. The six categories that the American College of Medical Geneticists recommend that causal diseases variants are placed include (1) disease-causing, (2) likely disease-causing, (3) possibly disease-causing, (4) likely not disease-causing, (5) not disease-causing and (6) variant of unknown clinical significance. Pathogenicity of genetic variants can be examined with bioinformatics prediction approaches, protein stability studies, transcriptional activity studies and allele- and/or gene-specific animal models. WES and WGS have low sensitivity and specificity. It has a high cost and fast turnaround time. However, classic genetic tests will continue to be used for high analytical sensitivity of specific defects and for the confirmation of genome findings
There remains no single test to detect the wide array of genetic defects that may be inherited or arise de novo; clinical diagnostics requires multiple approaches to determine a causal genetic defect. Genetic testing must be available globally through validated simple technologies for molecular diagnostics (such as direct PCR, linkage analysis or multiplex ligation-dependent probe amplification). As broader genomic information becomes available to providers and patients, partnerships will develop to convey patient-centered data, including incidental findings. The regulatory environment must adapt to the coming volume of genomic information to maximize benefit to patients and health-care systems and to match the expectations of the patient population with regard to these technologies.
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