Important and critical advances continued in the world of MDx with the invention of real-time PCR and its numerous variations, DNA microarray-based genotyping and transcription profiling, microbiome sequencing (gut and other areas of the body), proteomics (detection of disease-specific protein profiles), pharmacogenomics, nutrigenomics, forensic medicine and CRISPR/Cas9 genomic editing.Įven with the advent and explosion of diverse variant detection assays, DNA sequencing is the gold standard for identification and surveillance of pathogens. Rodney Rohde observes in the Bachelor of Clinical Laboratory Science Program, a NAACLS-accredited MLS 2-year undergraduate program at Texas State University.Īfter publication of the human genome draft sequence, the challenge to improve existing variant detection technologies to achieve robust, cost-effective, rapid and high-throughput analysis of genomic variation moved to the forefront of MDx. Robin Forbes performs a PCR assay to detect a methicillin-resistant Staphylococcus aureus (MRSA) gene as Dr. While many of these methods are now infrequently used in clinical microbiology, they paved the way to current, more sophisticated methods.
It helped establish 3 categories for variant detection, depending on the basis for discriminating the allelic variants: (1) enzymatic-based methods (e.g., RFLP and oligonucleotide ligation assay), (2) electrophoretic-based methods (e.g., single-strand conformation polymorphism (SSCP), heteroduplex analyses (HAD) and denaturing gradient gel electrophoresis) and (3) solid phase-based methods (e.g., reverse dot-blot and allele-specific hybridization). Not only did PCR bolster MDx in the clinical laboratory, it provided a foundation for the design and development of many variant detection schemes based on the amplification of DNA. With its powerful ability to exponentially amplify a target sequence, PCR allows the identification of a known mutation or sequence within hours. The development of PCR in the mid 1980s led to the golden era of molecular biology and MDx, and the use of a thermostable DNA polymerase from Thermus aquaticus (i.e., Taq polymerase, Saiki et al., 1988) quickly ushered this technique into the realm of laboratory medicine. This diagnostic, alongside the use of RFLP to characterize sickle cell alleles, set the foundation for characterization of other genetic diseases (e.g., cystic fibrosis), as well as infectious diseases, using MDx platforms. were the first to make a prenatal diagnosis of α-thalassemia using MDx techniques. DNA probes incorporating radioactive nucleotides allowed the analysis, via Southern blotting, of genomic regions, leading to the concept and application of restriction fragment length polymorphism (RFLP) to track variant alleles in the human genome. Sequencing and cDNA cloning were critical for establishing basic knowledge on the primary sequence of various genes. MDx grew from the early days of recombinant DNA technology.
While the ongoing SARS-CoV-2 / COVID-19 pandemic helped make molecular terms like ‘polymerase chain reaction’ (PCR), ‘false positive’ and ‘variant’ common, the field actually dates to 1949 with Linus Pauling and colleagues’ characterization of sickle cell anemia as a ‘molecular disease.’ However, it took decades for the scientific discipline of molecular biology to develop and become usable in the medical laboratory as a basis for disease diagnostics. Historical and Current Examples of Molecular Diagnostics For the first time in the history of the diagnostic laboratory, molecular pathology and diagnostics are extending the range of information available to physicians, pharmacists, geneticists, forensic scientists, research scientists and other healthcare professionals. The completion of the Human Genome Project and the rapid advancement of technology to arise out of that effort has moved from the research bench to the clinical laboratory bench with swift success. In the last decade or so, the clinical laboratory has seen an explosion in the available menu of tests based upon DNA and RNA analysis. Molecular diagnostic techniques and platforms are playing a larger and more critical role in all areas of anatomic and clinical pathology. Molecular diagnostics (MDx) is the rapidly developing area of laboratory medicine that investigates human, viral and microbial genomes and the products they encode.
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