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Home , Biomarker, Molecular diagnostics

The History and Evolution of

Molecular Diagnostics (MDx) is among Introduction Fast-forward to the mid-1800s the most highly complex disciplines in with Charles Darwin at the helm of Molecular Diagnostics is a branch of the modern science, a fact which has made evolutionary biology, promoting the industry, and involves the its development both long and arduous. idea that evolution was “descent with detection of genetic patterns in DNA The theoretical gestation of modification”. Then in 1866 Gregor and RNA, as well as patterns in spanned well over 2,000 years, from Johann Mendel, an Augustinian friar generation and usage. These detected classical Greek thinkers like Hippocrates whose pioneering work on the pea patterns (both genomic and proteomic) and Aristotle, all the way to the advent plant clearly showed documentable are used by scientists for classification of cellular biology in the 19th Century inheritance patterns. And although his and for the broadening of knowledge and through to modern times. work – which made use of breeding in connected fields, and by clinicians experiments – didn’t gain much traction Deoxyribonucleic Acid (DNA) and for aiding with diagnosis, prognosis and until it was re-discovered in 1900 by Ribonucleic Acid (RNA) were formally therapeutic monitoring. named in the first part of the 20th another team (Hugo de Vries, Carl The industry is a natural outgrowth Century, with the genetic coding Correns and Erich von Tschermak), by of both science and healthcare, and mechanism finally cracked in 1965. 1925 the model known as ‘Mendelian of medical technology and molecular The Human Project (HGP) inheritance’ was widely accepted. biology specifically. Uses of Molecular was begun by the US government in August Weismann’s contribution in Diagnostics are manifold, including 1987, during which time a multitude 1883 of his Germ Plasm theory was clinical , forensics testing, of scientists and clinicians began also instrumental in bringing about the epigenetics, immunotherapy and employing the knowledge that had coming sea change within scientific immunosuppression, metagenomics, thus far been gleaned for their own circles. molecular endocrinology, molecular particular uses. On the clinical side, Oxford physician , toxicology, personalized Sir Archibald Edward Garrod collected The Human Genome Project was medicine and more. completed in 2003, stimulating greater historical information in 1902 about Today the Molecular Diagnostics advances not only in DNA and RNA the family of one of his patients, industry is an exploding field with a sequencing, but in other areas of and concluded that his patient’s current market size of approximately Molecular Diagnostics. Simultaneous alkaptonuria was a recessive disorder $8B. advances in laboratory information passed down through inheritance. systems made it possible not only to Two Millennia of Inquiry This was quite possibly the first time store and copy the growing dataset, but in history that genetics – the idea that Before the rise of Molecular Diagnostics to use cutting-edge digital algorithms human can be inherited – was in the 1980s, clinicians were limited to to help extract the meaning within. discussed in a clinical setting. a generalized approach to diagnosing And advances in and health problems and for creating DNA and RNA metabolomics helped to increase our solutions for those problems. They In 1869 a Swiss physician named understanding of the biosciences in simply didn’t have any tools they could Friedrich Miescher analyzed the pus near equal measure. use to access the genetic information that was in the discarded bandages of The latest MDx technologies include of their patients, or for putting that one of his patients, and discovered that nanopore readers, one of which was information to use. But what they did the discharge contained microscopic recently used to read the Ebola virus have was access to human history, material. He called the substance genome in only 44 seconds. Such which itself is one of the greatest tools ‘nuclein’ because his microscope advances – combined with concurrent for stimulating innovation. allowed him to see that the substance advances in Computational Biology The history of genetics stretches back resided within the nucleus of the cells. and associated areas using software over two millennia to Hippocrates, who Then in 1878, Albrecht Kossel was the algorithms – are helping to dramatically is credited as being the first person to first scientist to isolate , reduce diagnostics times, allowing have speculated about inherited traits, and went on to isolate the five primary clinicians to treat their patients far more which he also suspected involved some nucleobases later in his career. quickly and efficiently. type of material transfer system in the At the beginning of the 20th Century the reproductive process.

1 History and Evolution of Molecular Diagnostics 11/2018 V1 terms DNA and RNA had not yet been experiment was inconclusive, Hershey Nirenberg began working on the enigma invented – but their existence was no went on to win the 1969 Nobel Prize – of DNA coding at the National Institute secret in microbiology circles. In those along with Max Delbrück and Salvador of Health (NIH). His beginning work early days DNA was known as ‘thymus Luria – for their work on viral genetics. was based on experiments in which he nucleic acid’, because the tissue that attempted to show the possibility of The Double-Helix and the Code of was being studied had been extracted RNA-triggered protein synthesis. Using from a thymus gland. In a similar way, Life the cytoplasm of E.Coli bacteria, he RNA was known as ‘yeast nucleic acid’, By the 1950s DNA and RNA were finally tested each of the 20 amino acids on with yeast as the source material. recognized as the containers of the the samples to find out which amino Phoebus Levene identified the genetic information in living systems. acid would be incorporated into the nucleotide unit of yeast nucleic acid in The double-helix model of DNA was final protein. 1909, carefully noting the base, provided by Francis Crick and James In 1961 Nirenberg’s team finally and phosphate components. Then in Watson in 1953, using X-ray diffraction discovered that phenylalanine was the 1929, while studying thymus nucleic imagery (with the famous ‘Photo amino acid which could be forced to acid, he identified deoxyribose sugar. 51’ taken by Raymond Gosling in the form the repeating protein chain with Leven’s ‘tetranucleotide hypothesis’ was previous year). Uracil. It was this experiment which the first to suggest that thymus nucleic In 1953, astronomer George Gamow proved to the team that the genetic acid was composed of four nucleotide created his ‘RNA Tie Club’, for which he code could be broken. units linked by the phosphate groups – hand-picked 20 scientists – a number Because Nirenberg was racing against although he thought that the ordering which matched the number of amino Nobel laureate Severo Ochoa to be the of the nucleotide units was the same in acids in the human body – and had first to crack the code of life, Nirenberg’s every cell. each of those scientists wear a tie which colleagues generously set down their It was Nikolai Koltsov who, in 1927, corresponded to a single amino acid. own experiments to assist in the effort. proposed his idea that hereditary traits His tie club provided Gamow with a way Ochoa decided to give up the race in were carried by two-stranded – or to show society his part in the race that ’62, because he felt that since Nirenberg “mirrored” – , with each was on at the time: who would be the had the lead, his own time would be strand acting as a genetic template. one to crack the genetic code. better spent on a different task. The very next year, Frederick Griffith In 1959, working separately, Marthe After four years of constant toil, with performed an experiment using two Gautier and Jérôme Lejeune each dozens of scientists scrambling about different but compatible bacterial discovered that Down’s syndrome was his lab, Nirenberg finally cracked the species, which provided the first caused by an additional gene in the DNA genetic code in 1965. real indication that DNA – as it was cluster – an extra copy of chromosome later known – was the true carrier of 21 (which is why the disorder was also A Chain Reaction hereditary information. called ‘trisomy 21’). This was absolutely During that same period, H. Gobind In 1944, Oswald Avery noticed that groundbreaking because it was the first Khorana was working on a way to when one pneumococcus species was time that chromosomes themselves completely synthesize a gene using intermingled with another, the first were being identified, using oligonucleotides. He developed multiple innocuous species suddenly took on the techniques such as Giemsa staining. techniques, some using oligonucleotides deadly characteristics of the second. He After the revolutionary discovery of as structural elements, and others called this the ‘transforming principle’. the double-helix formation of DNA by making use of oligonucleotides as Avery’s finds had a huge impact on Francis Crick and James Watson in 1953, templates and primers. Khorana was fellow researcher Erwin Chargaff, and in they postulated that the strand pairing awarded a Nobel Prize in 1968. 1950 Chargaff published his ‘Chargaff’s suggested the potential of being part of In 1971 Khorana’s team came up with Rules’ which, in part, stated that the a copying mechanism. This suggestion an idea they called ‘repair synthesis’. composition of DNA is subtly different won the pair a Nobel Prize in 1962, and This effort, developed as an attempt between different animal species. stimulated further research by others. to increase yield, is today seen as one The Hershey–Chase experiments, In 1957 Arthur Kornberg discovered the of the initial steps toward Polymerase conducted by Alfred Hershey and first DNA polymerase, which opened Chain Reaction (PCR) amplification, a Martha Chase in 1952, focused on the up the possibility of synthetic copying process that would become extremely virus Enterobacteria phage T2. The team of genetic material for the first time, instrumental in the eventual rise of discovered that because the DNA of and won him the Nobel Prize two years Molecular Diagnostics. the virus entered the host bacterium later in 1959. His work was incomplete, Although ‘repair synthesis’ was without the entry of much of the virus’ however, as much more needed to be incapable of achieving the exponential protein, that DNA was the likely genetic learned about the replication process. yield of PCR, it did illustrate that a transfer mechanism. Although the At around the same time, Marshall copy process was possible. At around

2 History and Evolution of Molecular Diagnostics 11/2018 V1 the same time, another member of increased the speed and accuracy of the and categorizing genetic material from Khorana’s team – Kjell Kleppe – wrote sequencing process. DNA samples). The scientists who a paper outlining an approach that This was followed by Dupont with their were involved knew that once this was strikingly similar to PCR, although Genesis 2000 which made use of a new streamlining could be accomplished, the his proposed process was never fully fluorescent labeling technique. ‘elucidation of the genome’ would come realized. to fruition much more quickly. The Human Genome Cetus Corporation – the company that By 2003 the NCHGR and their partners was eventually to own the rights to The first great leaps in modern announced that their goal of mapping the PCR process – was also founded genomics were finally accomplished the human genome was complete, in the same year (1971). They would – the knowledge of the coding and although it seemed likely to some eventually become the first Molecular mechanism, and the means of sample that this mapping would be continually Diagnostics company, with projects amplification. But without the deeper refined as time marched onward, the in the early ‘70s that included the knowledge of genetics – the knowledge existing mapping has been immensely development of diagnostic tests for of what the code was actually ‘saying’ – useful to the many scientists and genetic mutations. little progress could be made in the area clinicians who have made use of this that all this would eventually be used One key that was missing, however, immensely important database. for: health and medicine. was a polymerase that could withstand The First Uses of Molecular high temperatures. In 1976, Taq So the next race was on – the race to Diagnostics polymerase was successfully extracted crack the human genome. In 1991 the company Myriad was from Thermus aquaticus, a thermophilic In 1987 the United States Department bacterium. This polymerase could formed – one of the first of the of Energy (DOE) established the first Molecular Diagnostics companies. Their maintain stability at temperatures human genome project (HGP), in as high as 97.5 °C (with an optimum vision was to use genetic information to an effort to discover how to protect improve healthcare, and to do this they temperature range from 80 – 80 humans from the mutagenic effects of °C). This higher temperature range set out to develop the newest, cutting- radiation. The next year the NIH was edge diagnostic tools. opened up the door to new avenues in granted funding for a similar purpose, sequencing. and the two agencies soon signed a In 1996 Myriad announced the release Frederick Sanger put the Taq cooperation agreement (Memorandum of the first ever patient-level genetic polymerase discovery to good use, and of Understanding). In 1989 the name of testing system, BRACAnalysis. Detecting won the Nobel Prize in 1980 for his new the project was changed to the National the presence of a BRCA1 or BRCA2 method of DNA sequencing – known Center for Human Genome Research gene mutation, the test would provide as the chain-termination method, or (NCHGR). clinicians with a fast and efficient Sanger sequencing. This method greatly diagnosis for hereditary breast and The project would make extensive ovarian . For their innovation simplified the sequencing process, and use of technologies that were in was the most widely employed DNA Myriad was awarded the Laguna Niguel development at that time, which 1997 Best of Biotech Award. sequencing method for nearly four included PCR and capillary sequencing, decades after Sanger’s discovery (he restriction fragment-length In 1995 two different teams – actually made the discovery in 1976). polymorphisms (RFLP) and pulsed- Sooknanan and Malek, and Kacian and In 1983 – back at Cetus Corporation – field gel electrophoresis. Also included Fultz – were simultaneously developing Kary Mullis used his knowledge of the was the use of artificial chromosomes alternatives to PCR, with nucleic acid Sanger method as the foundation for a developed using yeast and bacteria. sequence-based amplification (NASBA), transcription mediated amplification new and revolutionary technique. He RFLP, developed in 1978 by David realized that a chain reaction could be (TMA) and self-sustained sequence Botstein et al, was an adopted replication (3SR). These techniques triggered by the repeated use of DNA technique that had been used by Ray polymerase – a chain reaction that relied on RNA polymerase rather than White on the Drosophila genome. DNA polymerase, to render a reverse would provide amplification of any DNA Botstein’s team felt that even with the segment. transcriptase that would produce DNA vast differences between fruit flies and from the RNA templates. PCR amplification was born – and with humans, attempting the technique with it, a whole new world of possibilities in human genes was an exciting prospect. Almost immediately, these innovations the world of genetics. RFLP would soon become a mainstay in made it possible to measure HIV-1 RNA genetic marker technology. in blood plasma, a test which became In 1987, Applied Biosystems (AB) the first widespread use of molecular unveiled the AB370, the first Much of the work during the early diagnostic technology. Having opened automatic sequencing machine. Their days of the HGP was in developing the door to viral load testing, kits soon revolutionary machine made use methods of streamlining the process of became available for the hepatitis C of capillary electrophoresis, which DNA sequencing (extracting, marking

3 History and Evolution of Molecular Diagnostics 11/2018 V1 virus (HCV), cytomegalovirus, Epstein- et al devised a system of embedding has been progressing ever since, and Barr virus and BK virus. genetic tissue in cold-temperature will undoubtedly continue far into the It was during these same years that plastic resin; this resin was the water- future. the diagnosis of herpes simplex virus miscible methyl methacrylate polymer In 2001 Dr. Bert Vogelstein of John (HSV)-associated encephalitis was aided known as Immunobed. Hopkins University was presented by the testing of HSV DNA in cerebral Also in 1999 a team led by Khuong the Award of Excellence by the AMP spinal fluid, as an alternative to brain Truong developed the technique known for his major contributions to the . Shortly thereafter a test for the as Fluorescence In Situ Hybridization understanding of genetics in connection detection of trachomatis (FISH) for the detection of lung with colorectal . In his acceptance was approved by the US Food and cancer. The technique used two-color speech, Vogelstein discussed genomic Drug Administration – their first-ever fluorescence on the long and short micro- and macro-instability, and studies approval of a molecular diagnostics arms of chromosome 3 to detect an involving gene expression changes in testing kit. imbalance. tumors. He also discussed his team’s Another innovation during the late In the late 90s the pharmaceutical use of ‘digital PCR’ which, although the 90s was the simultaneous detection industry recognized the need for term had been invented in the early and amplification of target nucleic simultaneous advances in diagnostic 90s, had seen a resurgence due to acid in real time. This process not tools on the clinical side of the equation other connected elements (software only eliminated the risk of carryover – advances which were already being enhancements, etc.). contamination, it also reduced the made by various individuals and teams. In the same year, Dr. Rudy Leibl of turnaround time to just a few hours. In 1999 the Association for Molecular Columbia University discussed the role In 1995, Craig Venter employed what Pathology (AMP) had co-founded The of genetics in the expression of was known as the ‘whole genome Journal of Medical Diagnostics, which and their effects on human body weight. provided an informational outlet for shotgun sequencing’ technique to Bioinformatics complete the Haemophilus specialists working in the field, and The It was also around the turn of the (bacterium) genome. Although the Journal of Molecular Diagnostics started millennium that advances in information shotgun sequencing technique was said the same year. technology – specifically bioinformatics by some to be unreliable – which is why The Millennium Turns – allowed huge productivity boosts for it was rejected for use by the HGP – it Although 2003 marked the official both scientists and clinicians. In 2000, nonetheless became quite popular in completion of the human genome Peter Cooper at the National Center for the genetics community. project, a bulk of the work had already Biotechnology Information began doing Back in 1993 the NCHGR had been accomplished by 2000. This workshops which taught specialists established their Division of Intramural provided the catalyst for an explosive how to use the web-based, sequence- Research (DIR). Three years later, eight environment of innovation within the manipulative genetics tools that his separate NIH divisions morphed into scientific community, fueled perhaps in organization had developed. Another a singular institute, the Center for part by the psychological thrust of the web-based tool, the GeneTests genetic Inherited Disease Research (CIDR), for turn of the millennium. Target-based test database – run by Bonnie Pagon at the study of genetic causes in relation drug discovery became a huge part of the University of Washington), allowed to disease. In 1997 the NCHGR was the R&D budgets of pharmaceutical instant access to tests – both scientific renamed the National Human Genome companies, as biochemists put the and clinical – from hundreds of labs Research Institute (NHGRI). estimated number of genes that could worldwide. be pharmacologically targeted at In 1996 the Health Insurance Portability In biochem labs around the globe, and Accountability Act (HIPAA) came between 3000 and 5000 (of the roughly Laboratory Information Management into effect, which – among other things 30,000 total genes in human DNA). But System (LIMS) and other specialized – made provisions for standardizing scientists eventually began to see that software were now making it possible to electronic health care (including this was likely a gross overestimate, work with immense datasets with much standards for electronic medical with only 500 targets discovered in greater ease, and allowed staggering records (EMR) and other electronic the ensuing five years of highly active computational analyses which were transactions), and established national research. theretofore impractical or impossible. identifiers for various entities in the The turn of the millennium also saw On the clinical side, physicians and lab medical establishment. an expansion of point-of-care testing technicians were finally able to take In 1999 the American Board of Medical devices and systems, which for the first advantage of digital means for working Genetics and the American Board of time allowed physicians and nurses with patient data using Electronic Pathology announced molecular genetic to use such technology directly at the Medical Record (EMR) systems as well pathology as a new joint subspecialty. location of their patients. This expanse as Laboratory Information System (LIS). In the same year, Sydney D. Finkelstein of point-of-care medical testing systems These IT advances reduced processing

4 History and Evolution of Molecular Diagnostics 11/2018 V1 times by eliminating the need for in a white paper of his from 1998. This which triggers an ion . juggling paper-based files, and by work formed the foundation of the next In 2005 Single- real-time speeding up the flow of information generation sequencing (NGS) process (SMRT) sequencing was developed by between specialists in disparate (aka ‘second generation process’ and Pacific Biosciences. SMRT made use of geographies using secure internet ‘high-throughput sequencing’). zero-mode waveguides (ZMWs) and connections. NGS was a groundbreaking innovation phospholinked nucleotides. SMRT is the It was at the turn of the millennia that that combined large-scale parallel most widely used sequencing platform Psyche Systems released The LabWeb sequencing, high throughput and today, even though it was developed System, a comprehensive Laboratory economy of operation. The platforms over a decade ago. Information System using web-based based on this approach utilized In 2007 another ‘sequencing by features like hyperlinked pages. The miniaturized and parallelized platforms synthesis’ method was created by system was highly advanced at the to process up to 600 million short reads Illumina/Solexa, which used modified time, and could incorporate many of DNA per 10-hour run. dNTPs containing a terminator which different multimedia types to enhance The new technique allowed – for blocks further polymerization. This the user experience. Psyche Systems the first time – an entire genome to meant that only a single base could now has eight state-of-the-art software be sequenced at once. This proved be added by a polymerase enzyme. packages in various areas of molecular useful not only with normal genome This type of reaction was carried out diagnostics, including Laboratory sequencing, but also with genome simultaneously across millions of Information Systems, reporting systems resequencing, profiling template molecules on a single surface, and connectivity solutions. (RNA-Seq), DNA-protein interactions making it highly parallelized. Another stimulator on the electronic (ChIP-sequencing), and epigenome Combinatorial probe-anchor synthesis side of medicine was the establishment characterization. (cPAS) sequencing used a combination of Health Level Seven (HL7), Employing new advancements of of sequencing by hybridization (SBH) which provided an international NGS, 454 Life Sciences (part of the and sequencing by ligation (SBL) standardization with digitally transferred 454 Corporation) released their new techniques on pools of probes, each data (clinical and administrative) in the Genome Sequencer 20 (GS20) machine probe consisting of an anchor sequence healthcare industry. This move implicitly in 2005. This was the very first complete and nine bases, where the interrogation required software companies to make NGS sequencer available on the open position was then dye-labeled, washed these standards a reality within the market, which caused an explosion and imaged. software code that so much of modern of productivity around the world, in a healthcare relies on. Sequencing by hybridization used micro- process which became known as 454 array technology – also known as DNA Quantitative PCR, or qPCR, was already pyrosequencing. Roche Diagnostics chips, ChIP sequencing, and bio-chips – in development in the 90s as an update acquired 454 Life Sciences in 2007. which contain up to billions of synthetic to traditional PCR. With the production NGS machines found use in myriad oligonucleotides on a single surface. of complementary RT-PCR using an fields, including metagenomics, Sequencing by ligation (SOLiD initial reverse transcription (RT) step, molecular endocrinology, amplification of any type of RNA could sequencing) determines the underlying anatomic pathology, cytogenetics sequence of nucleotides in a given now be accomplished. qPCR quickly testing, immunosuppression and became the new standard in the DNA sequence by making use of the immunotherapy, , mismatch sensitivity of DNA ligase. biological toolbox. and evolutionary biology. In 2001 Jules Meijerink et al discovered Polony sequencing, developed by Other Sequencing Technologies a common problem in clinical settings, George M. Church at Harvard, used in which patient samples and calibration Many newer high-throughput emulsion PCR, an in vitro paired-tag samples showed unequal efficiencies sequencing (HTS) methods were library, ligation-based sequencing during amplification (typically due developed during this period. chemistry and an automated microscope. This process was used to contamination with various In 2002 Ion semiconductor sequencing to sequence a full E. coli genome in compounds that were common to the (aka Ion Torrent sequencing) made use 2005, with an accuracy greater than qPCR process). They developed the of the detection of hydrogen ions that 99.9999%. efficiency compensation control (ECC) to are released during the polymerization compensate for the problem. of DNA. This was one of the ‘sequencing DNA nanoball sequencing, developed by Next Generation Sequencing by synthesis’ methods, in which dNTP Complete Genomics, employed rolling is flooded into a microwell containing circle replication for the amplification In 2000, Jonathan Rothberg founded the DNA sample. If the dNTP is of genetic material into nanoballs. The 454 Corporation as a subsidiary of complementary to the leading template sequence was then determined using Curagen. His ideas were based on work nucleotide, hydrogen ions are released, unchained sequencing by ligation. done by P. Mayer et al that was outlined

5 History and Evolution of Molecular Diagnostics 11/2018 V1 Heliscope single molecule sequencing The aforementioned ABI 310, which was a patient rather than in a laboratory was introduced by Helicos Biosciences. a single channel CE system, and the ABI environment. But it wasn’t until the This solution added poly-A tail adapters 3100, ABI’s multi-channel system, made 2000s that POC technology would begin to DNA fragments, and followed this it possible to quickly test for fragile X to include for molecular step with extension-based sequencing syndrome and for HIV genotyping. diagnostics. and cyclic washes with fluorescently Sequence-based diagnostics (SBD) In 2003, following the biological threat labeled nucleotides. Heliscope were routinely used to assess HIV of Anthrax at the United States Postal performed the reading. . CE systems became Service, the USPS awarded a $175M Concurrent Advances in Molecular the leader on this front within the contract to Northrop Grumman Corp., Diagnostics clinical environment, making it possible in cooperation with the company for clinicians to work with the HIV Cepheid. The result of the contract was Total growth in the Molecular genome, in addition to facilitating the Biohazard Detection System (BDS), Diagnostics industry from 2000 to 2005 the identification of yeast and fungal which began national rollout at USPS was between 10% and 20%, and most using 16S and 26S rRNA centers in 2004. of this growth was directly stimulated identification. by advances in both sequencing This system, called GeneXpert, could technologies and in bioinformatics By 2005 hardware and software perform real-time PCR on a sample software technologies. These platforms had become available for within 30 minutes, by users who had no simultaneous advances made it possible applications as diverse as forensic need for biochem training. This system to automate the sequencing process, testing (including forensic toxicology), marked the birth of POC for molecular freeing up time for scientists to focus paternity, immigration, genetic diagnostics. on data analysis. And the new software and infectious disease, molecular Other companies working on POC tools allowed analysis to be done with endocrinology, molecular oncology, molecular diagnostics equipment in greater speed and efficiency, allowing evolutionary biology and anthropology, 2005 were IQuum, Nanosphere, Inc. scientists to query databases in more prenatal diagnostics, and the genetic and Nanogen Inc. in the U.S., Enigma specific ways. modification of food crops. Diagnostics, LGC and Lumora Ltd. in For the first time in history, hospitals In 2007, Applied Biosystems (now Life England, and IMM in Germany. could perform genetic testing on a STAT Technologies) released their SOLiD In 2006, Roche introduced LightCycler basis, a fact which greatly improved the platform which made use of next- SeptiFast, a kit which could rapidly environment of healthcare. This was an generation sequencing technology to detect the cause of , by analyzing important step in the rise of molecular deliver up to 4 billion bases of sequence the genetics of microbial agents in the medicine. data per run. This made the SOLiD blood. Up to 25 different microbial platform the highest-throughput system Back in 2001, cooperation between agents could be discovered using the kit, of its day, and with a raw base accuracy which proved invaluable for hospitals the NIH, the American College of greater than 99.94%. Medical Genetics (ACMG) and the around the world. American College of Obstetricians and Although multiplex-PCR was conceived By 2008 companies such as TwistDX Gynecologists (ACOG) gave rise to a in 1988 for the detection of genetic were using Recombinase Polymerase test for (CF), deletions, its use increased significantly Amplification (RPA) isothermal which was strongly advised for pregnant in the 2000s in the wake of better technology to bring genetic technology women. Assay platforms for the test and faster assay processing. In 2008, out of the lab and directly to the included Innogenetics CFTR33 LiPA multiplex-PCR found new uses, for patient. Although POC was already a (Alpharetta, GA), Roche CF Gold, and instance with the analysis of SNPs term used for many years, devices were the CF v3 OLA assay (Abbott-Celera, (single-nucleotide polymorphisms) becoming smaller, more robust and Abbott Park, IL), together with their and (tracts of repetitive more accurate as time progressed. associated reagent kits (ASRs). DNA). In 2009, funding for POC molecular As assay automation came to the fore Before the 2000s, diagnostics testing exploded, and by during this period, sample processing (MS) was only employed for protein 2013 $650M had been secured in the was still an issue. Thermal Systems detection and characterization. Since industry. But even with such funding, responded to the problem with the the turn of the millennium, MS has been manufacturers at the time noted that Kingfisher, a smaller nucleic acid found to be highly useful for microbial although hospitals had readily adopted extraction instrument. The Tecan and viral detection. such test kits, adoption in clinics was provided an innovative platform with Point of Care Diagnostics dismal. its use of robotics for liquid handling. Roche, MagnaPure and Qiagen Point of Care (POC) diagnostics was a In 2011 David M. Pearce et al developed were soon to follow with their new term invented in the 1980’s, and refers a new electrochemical method of automated extractors. to any test that can be performed near detecting Chlamydia trachomatis, and

6 History and Evolution of Molecular Diagnostics 11/2018 V1 by 2015 his team’s technique was put to In 2015 the BGISEQ-500 sequencer was genetic mapping, advanced sequence use by Atlas Genetics in a POC kit called released by BGI, integrating automated analysis, protein classification and long- io System. sample preparation, sequencing and time simulation of protein molecules, Newer POC systems include those by data analysis in an all-in-one machine fold prediction, structure comparison Vantix Diagnostics, the i-STAT Alinity that would fit on a desktop. The algorithms, and comparative genomics. system, Pandora’s CDx system, and machine additionally made use of Radio Other computer experts are working Luminex and their xTAG systems (8 Frequency Identification (RFID), as well in areas such as query optimization different kits). And leading the pack is as barcode scanning technologies, for and text mining. Query optimization Oxford Nanopore Technologies with its further streamlining of genetic projects. allows MDx specialists more flexibility MinION device, allowing DNA and RNA In 2016 the machine was offered with a and power when they interface with sequencing in real-time, using a USB brand new thermoplastic called PEEK. datasets using databases such as SQL. device that weighs less than 100g. BGI’s machine employs DNA Nanoballs And text mining brings in the immense (DNB) technology, which uses rolling- power of Google, allowing the entire Newer Technologies circle replication, as well as Probe- internet to be leveraged for the It’s an interesting fact that as MDx Anchor Synthesis (cPAS) technology. acquisition of subjective associations processes are being continually sped In 2016 Illumina released their HiSeq using unsupervised machine learning up by various means, including digital 4000 Sequencing System, which uses and natural language analysis. analytics and the use of robotics, older patterned flow-cell technology which One of the most explosive and technologies have gotten a booster allows users to sequence “12 , important fields in MDx is shot and brought back up to the fore – 100 , or 96 exomes in discovery and validation, and many only in a slightly different context. One fewer than 3.5 days”. experts suggest that this is the key to good example of this phenomenon is progress. and multiplex PCR, a process that makes The newest sequencing technologies – validation will continue for many, many use of standard PCR, but creates called 3rd Generation Sequencing – can years, with each successive biomarker a parallelized system within which read over 10,000 base pairs, or map finding important use in hospitals and many PCR assays (and their associated over 100,000 base pair molecules. clinics around the world, and each one processes) are occurring simultaneously, Nanopore Sequencing has made it adding to the total body of knowledge. allowing much more complex analyses possible to sequence single molecules as a result. of DNA and RNA without the need One such discovery is the use of Single-Nucleotide Polymorphisms On the other hand, brand new for amplification. The process also (SNP) as . Recent genome- discoveries are being made more and eliminates the need for chemical wide association studies were done more often, with the time shrinking labeling. In this process, electrophoresis in China on various populations, and between each new innovation, making is used to transport a sample through it was discovered that SNP loci in five modern tech-news a highly dense web a micro-orifice with a diameter of 1 different genes were associated with of explosive modernization. This – the nanometer. Sequencing is facilitated susceptibility to esophageal squamous last section of this article – will focus because of differences in electric cell carcinoma (ESCC). Much more on discoveries that have been made in current densities caused by different work is being done by many different the last few years, including the newest genetic samples, which affects the flow teams around the world using SNP’s as and greatest, bleeding-edge innovations through the nanopore channel. biomarkers. that have helped in making MDx what The aforementioned MinION, by Oxford it is today. Nanopore Technologies, puts nanopore In the last 5 years, nearly 1000 papers were published on the subject of By 2012, Luminex had on offering their sequencing to highly effective use. Their biomarker discovery through the xTAG range of testing kits, including the two larger systems – the benchtop use of metabolomics, and in that xTAG Nucleic Acid Assay Technology, GridION and the higher-thoughput, same time many such biomarkers xTAG Cystic Fibrosis, xTAG Respiratory higher-sample number PromethION – have been validated. This vindicates Viral Panel, xTAG Gastrointestinal also employ nanopore technology. metabolomics as a highly useful tool in Panel, xTAG CYP2D6 Kit, xTAG Another cutting-edge area of research biomarker research, with its use of mass CYP2C19 Kit. are those of Computational Biology, spectrometry and data-mining software. Around the same time, the eSensor Statistical Genetics and Mathematical A recent advancement in bioinformatics respiratory viral panel was introduced Modeling, each of which use statistical is The BioCompute Object (BCO) by GenMark Diagnostics. These new kits methods for extracting meaning from Project, which began as a collaborative provided clinicians with more amazing genetic datasets. Dr. Fengzhu Sun is one effort between the FDA and George tools to stick into their belts – so-to- such specialist in these innovative fields. Washington University. It was instigated speak – decreasing time for assays and He and similar specialists are using as a response to the challenge of analytics for their patients. computers and algorithmic processing to assist with MDx tasks as diverse as sharing scientific workflows and

7 History and Evolution of Molecular Diagnostics 11/2018 V1 documentation by different teams, not needed prescription to their patient’s only for the sake of peer reviews but local pharmacy on the same day. perhaps more importantly to allow And on the proteomics front, data- information sharing itself to be the independent acquisition (DIA) is catalyst for the evolution of science. the new technology on the block. The BCO Project now includes over 20 Although the wrinkles have not been universities, biotechnology companies, completely ironed out, DIA looks to pharmaceutical companies, and public/ be quite promising. Whereas data- private partnerships. dependent acquisition (DDA) has Other recent MDx advances include been the paradigm for a decade and work on miRNA and long non-coding a half, in combination with liquid RNA, Genetic testing for high-grade -tandem mass osteosarcoma, molecular and spectrometry (LC-MS/MS) and multiple point-of-care diagnostics for Ebola, reaction monitoring (MRM), DIA biomarkers for viral hepatitis samples every peptide in a protein and hepatocellular carcinoma, digest, comprehensively and repeatedly, electrochemical immunoassay for producing a complex set of mass tumor markers based on hydrogels, spectra at the output of the process. effective early detection of oral cancer The spectral output is interpreted using using a simple and inexpensive point external spectral libraries derived from of care device in oral rinses, and DNA prior DIA experiments or from auxiliary . DDA data, producing identification Antibiotic resistance poses a serious and quantification nearly equal to that threat to humanity, and as such it will achieved by MRM/PRM. Cutting-edge be a theme that is likely to impact the bioinformatics techniques are being MDx space for some time. It has been introduced to address the remaining suggested that antibiotic resistance will issues with DIA, and the potential take over cancer as the leading cause of results are quite promising. death by 2050. Conclusion The rise of antibiotic resistance can The future is always bright wherever be attributed to over-prescription of it features the minds of intellectually- antibiotics, in a healthcare system full of driven men and women, who are ever doctors who – being at a technical loss in pursuit of that ideal of perfection because they cannot quickly discover which has been the dream of mankind the exact cause of or sickness since time immemorial. In the realm of – will prescribe antibiotics. This problem MDx, physically diminutive POC devices is exacerbated by the public belief in with ever-increasing power and scope antibiotics as a ‘cure-all’ (because the will always be the goal for clinicians average person doesn’t understand the – and for the armies of scientists and complexities of biology and medicine). technicians who build those clinicians’ MDx is now coming to the aid, not tools. This ideal is perfectly exemplified directly but indirectly, by dramatically by the fictitious and oft-alluded-to reducing the time it takes to pinpoint tricorder, as seen in myriad episodes the cause of infection, via the detection of Star Trek. And although many of the specific microbial agent. New specialists see this allusion as trite, such systems are allowing a patient’s sample a handheld device is slowly but surely in to be loaded into a cartridge and placed the process of being manifested by the into a process that will test for hundreds legions of scientists and great thinkers of different at once, as of our great modern age. well as testing for antibiotic resistance markers. Using such systems, current state-of-the-art technology brings analytical results down to approximately five hours, which is a great boon to physicians who can then send the

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