Molecular diagnostics

Last updated

Specialist using "QIAsymphony", an automation platform for molecular diagnostic tests Molecular diagnostics qia symphony.jpg
Specialist using "QIAsymphony", an automation platform for molecular diagnostic tests

Molecular diagnostics is a collection of techniques used to analyze biological markers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing. In medicine the technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients, [1] [2] : foreword  and in agricultural biosecurity similarly to monitor crop- and livestock disease, estimate risk, and decide what quarantine measures must be taken. [3]

Contents

By analysing the specifics of the patient and their disease, molecular diagnostics offers the prospect of personalised medicine. [4] These tests are useful in a range of medical specialties, including infectious disease, oncology, human leucocyte antigen typing (which investigates and predicts immune function), coagulation, and pharmacogenomics —the genetic prediction of which drugs will work best. [5] : v-vii  They overlap with clinical chemistry (medical tests on bodily fluids).

History

Molecular diagnostics uses techniques such as mass spectrometry and gene chips to capture the expression patterns of genes and proteins Molecular Diagnostics.jpg
Molecular diagnostics uses techniques such as mass spectrometry and gene chips to capture the expression patterns of genes and proteins

The field of molecular biology grew in the late twentieth century, as did its clinical application. In 1980, Yuet Wai Kan et al. suggested a prenatal genetic test for Thalassemia that did not rely upon DNA sequencing —then in its infancy—but on restriction enzymes that cut DNA where they recognised specific short sequences, creating different lengths of DNA strand depending on which allele (genetic variation) the fetus possessed. [6] In the 1980s, the phrase was used in the names of companies such as Molecular Diagnostics Incorporated [7] and Bethseda Research Laboratories Molecular Diagnostics. [8] [9]

During the 1990s, the identification of newly discovered genes and new techniques for DNA sequencing led to the appearance of a distinct field of molecular and genomic laboratory medicine; in 1995, the Association for Molecular Pathology (AMP) was formed to give it structure. In 1999, the AMP co-founded The Journal of Medical Diagnostics. [10] Informa Healthcare launched Expert Reviews in Medical Diagnostics in 2001. [1] From 2002 onwards, the HapMap Project aggregated information on the one-letter genetic differences that recur in the human population—the single nucleotide polymorphisms —and their relationship with disease. [2] : ch 37  In 2012, molecular diagnostic techniques for Thalassemia use genetic hybridization tests to identify the specific single nucleotide polymorphism causing an individual's disease. [11]

As the commercial application of molecular diagnostics has become more important, so has the debate about patenting of the genetic discoveries at its heart. In 1998, the European Union's Directive 98/44/ECclarified that patents on DNA sequences were allowable. [12] In 2010 in the US, AMP sued Myriad Genetics to challenge the latter's patents regarding two genes, BRCA1, BRCA2, which are associated with breast cancer. In 2013, the U.S. Supreme Court partially agreed, ruling that a naturally occurring gene sequence could not be patented. [13] [14]

Techniques

The Affymetrix 5.0, a microarray chip Affymetrix 5.0 microarray.jpg
The Affymetrix 5.0, a microarray chip

Development from research tools

The industrialisation of molecular biology assay tools has made it practical to use them in clinics. [2] : foreword  Miniaturisation into a single handheld device can bring medical diagnostics into the clinic and into the office or home. [2] : foreword  The clinical laboratory requires high standards of reliability; diagnostics may require accreditation or fall under medical device regulations. [15] As of 2011, some US clinical laboratories nevertheless used assays sold for "research use only". [16]

Laboratory processes need to adhere to regulations, such as the Clinical Laboratory Improvement Amendments, Health Insurance Portability and Accountability Act, Good Laboratory Practice, and Food and Drug Administration specifications in the United States. Laboratory Information Management Systems help by tracking these processes. [17] Regulation applies to both staff and supplies. As of 2012, twelve US states require molecular pathologists to be licensed; several boards such as the American Board of Medical Genetics and the American Board of Pathology certify technologists, supervisors, and laboratory directors. [18]

Automation and sample barcoding maximise throughput and reduce the possibility of error or contamination during manual handling and results reporting. Single devices to do the assay from beginning to end are now available. [15]

Assays

Molecular diagnostics uses in vitro biological assays such as PCR-ELISA or Fluorescence in situ hybridization. [19] [20] The assay detects a molecule, often in low concentrations, that is a marker of disease or risk in a sample taken from a patient. Preservation of the sample before analysis is critical. Manual handling should be minimised. [21] The fragile RNA molecule poses certain challenges. As part of the cellular process of expressing genes as proteins, it offers a measure of gene expression but it is vulnerable to hydrolysis and breakdown by ever-present RNAse enzymes. Samples can be snap-frozen in liquid nitrogen or incubated in preservation agents. [2] : ch 39 

Because molecular diagnostics methods can detect sensitive markers, these tests are less intrusive than a traditional biopsy. For example, because cell-free nucleic acids exist in human plasma, a simple blood sample can be enough to sample genetic information from tumours, transplants or an unborn fetus. [2] : ch 45  Many, but not all, molecular diagnostics methods based on nucleic acids detection use polymerase chain reaction (PCR) to vastly increase the number of nucleic acid molecules, thereby amplifying the target sequence(s) in the patient sample. [2] : foreword  PCR is a method that a template DNA is amplified using synthetic primers, a DNA polymerase, and dNTPs. The mixture is cycled between at least 2 temperatures: a high temperature for denaturing double-stranded DNA into single-stranded molecules and a low temperature for the primer to hybridize to the template and for the polymerase to extend the primer. Each temperature cycle theoretically doubles the quantity of target sequence. Detection of sequence variations using PCR typically involves the design and use oligonucleotide reagents that amplify the variant of interest more efficiently than wildtype sequence. PCR is currently the most widely used method for detection of DNA sequences. [22] The detection of the marker might use real time PCR, direct sequencing, [2] : ch 17  microarray chips —prefabricated chips that test many markers at once, [2] : ch 24  or MALDI-TOF [23] The same principle applies to the proteome and the genome. High-throughput protein arrays can use complementary DNA or antibodies to bind and hence can detect many different proteins in parallel. [24] Molecular diagnostic tests vary widely in sensitivity, turn around time, cost, coverage and regulatory approval. They also vary in the level of validation applied in the laboratories using them. Hence, robust local validation in accordance with the regulatory requirements and use of appropriate controls is required especially where the result may be used to inform a patient treatment decision. [25]

Benefits

A microarray chip contains complementary DNA (cDNA) to many sequences of interest. The cDNA fluoresces when it hybridises with a matching DNA fragment in the sample. Microarray Comparative Genomic Hybridisation.jpg
A microarray chip contains complementary DNA (cDNA) to many sequences of interest. The cDNA fluoresces when it hybridises with a matching DNA fragment in the sample.

Prenatal

Conventional prenatal tests for chromosomal abnormalities such as Down Syndrome rely on analysing the number and appearance of the chromosomes—the karyotype. Molecular diagnostics tests such as microarray comparative genomic hybridisation test a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive, but as of 2013 it is still an adjunct to the conventional tests. [26]

Treatment

Some of a patient's single nucleotide polymorphisms —slight differences in their DNA—can help predict how quickly they will metabolise particular drugs; this is called pharmacogenomics. [27] For example, the enzyme CYP2C19 metabolises several drugs, such as the anti-clotting agent Clopidogrel, into their active forms. Some patients possess polymorphisms in specific places on the 2C19 gene that make poor metabolisers of those drugs; physicians can test for these polymorphisms and find out whether the drugs will be fully effective for that patient. [28] Advances in molecular biology have helped show that some syndromes that were previously classed as a single disease are actually multiple subtypes with entirely different causes and treatments. Molecular diagnostics can help diagnose the subtype—for example of infections and cancers—or the genetic analysis of a disease with an inherited component, such as Silver-Russell syndrome. [1] [29]

Infectious disease

Molecular diagnostics are used to identify infectious diseases such as chlamydia, [30] influenza virus [31] and tuberculosis; [32] or specific strains such as H1N1 virus [33] or SARS-CoV-2. [34] Genetic identification can be swift; for example a loop-mediated isothermal amplification test diagnoses the malaria parasite and is rugged enough for developing countries. [35] But despite these advances in genome analysis, in 2013 infections are still more often identified by other means—their proteome, bacteriophage, or chromatographic profile. [36] Molecular diagnostics are also used to understand the specific strain of the pathogen—for example by detecting which drug resistance genes it possesses—and hence which therapies to avoid. [36] In addition, assays based on metagenomic next generation sequencing can be implemented to identify pathogenic organisms without bias. [37]

Disease risk management

A patient's genome may include an inherited or random mutation which affects the probability of developing a disease in the future. [27] For example, Lynch syndrome is a genetic disease that predisposes patients to colorectal and other cancers; early detection can lead to close monitoring that improves the patient's chances of a good outcome. [38] Cardiovascular risk is indicated by biological markers and screening can measure the risk that a child will be born with a genetic disease such as Cystic fibrosis. [39] Genetic testing is ethically complex: patients may not want the stress of knowing their risk. [40] In countries without universal healthcare, a known risk may raise insurance premiums. [41]

Cancer

Cancer is a change in the cellular processes that cause a tumour to grow out of control. [27] Cancerous cells sometimes have mutations in oncogenes, such as KRAS and CTNNB1 (β-catenin). [42] Analysing the molecular signature of cancerous cells—the DNA and its levels of expression via messenger RNA —enables physicians to characterise the cancer and to choose the best therapy for their patients. [27] As of 2010, assays that incorporate an array of antibodies against specific protein marker molecules are an emerging technology; there are hopes for these multiplex assays that could measure many markers at once. [43] Other potential future biomarkers include micro RNA molecules, which cancerous cells express more of than healthy ones. [44]

Cancer is a disease with excessive molecular causes and constant evolution. There's also heterogeneity of disease even in an individual. Molecular studies of cancer have proved the significance of driver mutations in the growth and metastasis of tumors. [45] Many technologies for detection of sequence variations have been developed for cancer research. These technologies generally can be grouped into three approaches: polymerase chain reaction (PCR), hybridization, and next-generation sequencing (NGS). [22] Currently, a lot of PCR and hybridization assays have been approved by FDA as in vitro diagnostics. [46] NGS assays, however, are still at an early stage in clinical diagnostics. [47]

To do the molecular diagnostic test for cancer, one of the significant issue is the DNA sequence variation detection. Tumor biopsy samples used for diagnostics always contain as little as 5% of the target variant as compared to wildtype sequence. Also, for noninvasive applications from peripheral blood or urine, the DNA test must be specific enough to detect mutations at variant allele frequencies of less than 0.1%. [22]

Currently, by optimizing the traditional PCR, there's a new invention, amplification-refractory mutation system (ARMS) is a method for detecting DNA sequence variants in cancer. The principle behind ARMS is that the enzymatic extension activity of DNA polymerases is highly sensitive to mismatches near the 3' end of primer. [22] Many different companies have developed diagnostics tests based on ARMS PCR primers. For instance, Qiagen therascreen, [48] Roche cobas [49] and Biomerieux THxID [50] have developed FDA approved PCR tests for detecting lung, colon cancer and metastatic melanoma mutations in the KRAS, EGFR and BRAF genes. Their IVD kits were basically validated on genomic DNA extracted from FFPE tissue.

There are also microarrays that utilize hybridization mechanism to diagnose cancer. More than a million of different probes can be synthesized on an array with Affymetrix's Genechip technology with a detection limit of one to ten copies of mRNA per well. [22] Optimized microarrays are typically considered to produce repeatable relative quantitation of different targets. [51] Currently, FDA have already approved a number of diagnostics assays utilizing microarrays: Agendia's MammaPrint assays can inform the breast cancer recurrence risk by profiling the expression of 70 genes related to breast cancer; [52] Autogenomics INFNITI CYP2C19 assay can profile genetic polymorphisms, whose impacts on therapeutic response to antidepressants are great; [53] and Affymetrix's CytoScan Dx can evaluate intellectual disabilities and congenital disorders by analyzing chromosomal mutation. [54]

In the future, the diagnostic tools for cancer will likely to focus on the Next Generation Sequencing (NGS). By utilizing DNA and RNA sequencing to do cancer diagnostics, technology in the field of molecular diagnostics tools will develop better. Although NGS throughput and price have dramatically been reduced over the past 10 years by roughly 100-fold, we remain at least 6 orders of magnitude away from performing deep sequencing at a whole genome level. [22] Currently, Ion Torrent developed some NGS panels based on translational AmpliSeq, for example, the Oncomine Comprehensive Assay. [55] They are focusing on utilizing deep sequencing of cancer-related genes to detect rare sequence variants.

Molecular diagnostics tool can be used for cancer risk assessment. For example, the BRCA1/2 test by Myriad Genetics assesses women for lifetime risk of breast cancer. [22] Also, some cancers are not always employed with clear symptoms. It is useful to analyze people when they do not show obvious symptoms and thus can detect cancer at early stages. For example, the ColoGuard test may be used to screen people over 55 years old for colorectal cancer. [56] Cancer is a longtime-scale disease with various progression steps, molecular diagnostics tools can be used for prognosis of cancer progression. For example, the OncoType Dx test by Genomic Health can estimate risk of breast cancer. Their technology can inform patients to seek chemotherapy when necessary by examining the RNA expression levels in breast cancer biopsy tissue. [57]

With rising government support in DNA molecular diagnostics, it is expected that an increasing number of clinical DNA detection assays for cancers will become available soon. Currently, research in cancer diagnostics are developing fast with goals for lower cost, less time consumption and simpler methods for doctors and patients.

See also

Related Research Articles

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.

Noninvasive genotyping is a modern technique for obtaining DNA for genotyping that is characterized by the indirect sampling of specimen, not requiring harm to, handling of, or even the presence of the organism of interest. Beginning in the early 1990s, with the advent of PCR, researchers have been able to obtain high-quality DNA samples from small quantities of hair, feathers, scales, or excrement. These noninvasive samples are an improvement over older allozyme and DNA sampling techniques that often required larger samples of tissue or the destruction of the studied organism. Noninvasive genotyping is widely utilized in conservation efforts, where capture and sampling may be difficult or disruptive to behavior. Additionally, in medicine, this technique is being applied in humans for the diagnosis of genetic disease and early detection of tumors. In this context, invasivity takes on a separate definition where noninvasive sampling also includes simple blood samples.

Molecular pathology is an emerging discipline within pathology which is focused in the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids. Molecular pathology shares some aspects of practice with both anatomic pathology and clinical pathology, molecular biology, biochemistry, proteomics and genetics, and is sometimes considered a "crossover" discipline. It is multi-disciplinary in nature and focuses mainly on the sub-microscopic aspects of disease. A key consideration is that more accurate diagnosis is possible when the diagnosis is based on both the morphologic changes in tissues and on molecular testing.

Digital polymerase chain reaction is a biotechnological refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids strands including DNA, cDNA, or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring nucleic acids amounts, with the former being a more precise method than PCR, though also more prone to error in the hands of inexperienced users. A "digital" measurement quantitatively and discretely measures a certain variable, whereas an “analog” measurement extrapolates certain measurements based on measured patterns. PCR carries out one reaction per single sample. dPCR also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The method has been demonstrated as useful for studying variations in gene sequences — such as copy number variants and point mutations — and it is routinely used for clonal amplification of samples for next-generation sequencing.

Minimal residual disease (MRD), also known as Molecular residual disease, is the name given to small numbers of cancer cells that remain in a person either during or after treatment when the patient is in remission. Sensitive molecular tests are either in development or available to test for MRD. These can measure minute levels of cancer cells in tissue samples, sometimes as low as one cancer cell in a million normal cells, either using DNA, RNA or proteins.

High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah. It has advantages over other genotyping technologies, namely:

The Xpert MTB/RIF is a cartridge-based nucleic acid amplification test (NAAT) for simultaneous rapid tuberculosis diagnosis and rapid antibiotic sensitivity test. It is an automated diagnostic test that can identify Mycobacterium tuberculosis (MTB) DNA and resistance to rifampicin (RIF). It was co-developed by the laboratory of Professor David Alland at the University of Medicine and Dentistry of New Jersey (UMDNJ), Cepheid Inc. and Foundation for Innovative New Diagnostics, with additional financial support from the US National Institutes of Health (NIH).

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

COLD-PCR is a modified polymerase chain reaction (PCR) protocol that enriches variant alleles from a mixture of wildtype and mutation-containing DNA. The ability to preferentially amplify and identify minority alleles and low-level somatic DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. Detection of mutations is important in the case of early cancer detection from tissue biopsies and body fluids such as blood plasma or serum, assessment of residual disease after surgery or chemotherapy, disease staging and molecular profiling for prognosis or tailoring therapy to individual patients, and monitoring of therapy outcome and cancer remission or relapse. Common PCR will amplify both the major (wildtype) and minor (mutant) alleles with the same efficiency, occluding the ability to easily detect the presence of low-level mutations. The capacity to detect a mutation in a mixture of variant/wildtype DNA is valuable because this mixture of variant DNAs can occur when provided with a heterogeneous sample – as is often the case with cancer biopsies. Currently, traditional PCR is used in tandem with a number of different downstream assays for genotyping or the detection of somatic mutations. These can include the use of amplified DNA for RFLP analysis, MALDI-TOF genotyping, or direct sequencing for detection of mutations by Sanger sequencing or pyrosequencing. Replacing traditional PCR with COLD-PCR for these downstream assays will increase the reliability in detecting mutations from mixed samples, including tumors and body fluids.

Natera, Inc. is a clinical genetic testing company based in Austin, Texas that specializes in non-invasive, cell-free DNA (cfDNA) testing technology, with a focus on women’s health, cancer, and organ health. Natera’s proprietary technology combines novel molecular biology techniques with a suite of bioinformatics software that allows detection down to a single molecule in a tube of blood. Natera operates CAP-accredited laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) in San Carlos, California and Austin, Texas.

Cell-free fetal DNA (cffDNA) is fetal DNA that circulates freely in the maternal blood. Maternal blood is sampled by venipuncture. Analysis of cffDNA is a method of non-invasive prenatal diagnosis frequently ordered for pregnant women of advanced maternal age. Two hours after delivery, cffDNA is no longer detectable in maternal blood.

<span class="mw-page-title-main">Circulating tumor DNA</span> Tumor-derived fragmented DNA in the bloodstream

Circulating tumor DNA (ctDNA) is tumor-derived fragmented DNA in the bloodstream that is not associated with cells. ctDNA should not be confused with cell-free DNA (cfDNA), a broader term which describes DNA that is freely circulating in the bloodstream, but is not necessarily of tumor origin. Because ctDNA may reflect the entire tumor genome, it has gained traction for its potential clinical utility; "liquid biopsies" in the form of blood draws may be taken at various time points to monitor tumor progression throughout the treatment regimen.

A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. Like traditional biopsy, this type of technique is mainly used as a diagnostic and monitoring tool for diseases such as cancer, with the added benefit of being largely non-invasive. Liquid biopsies may also be used to validate the efficiency of a cancer treatment drug by taking multiple samples in the span of a few weeks. The technology may also prove beneficial for patients after treatment to monitor relapse.

<span class="mw-page-title-main">Surveyor nuclease assay</span>

Surveyor nuclease assay is an enzyme mismatch cleavage assay used to detect single base mismatches or small insertions or deletions (indels).

CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) is a next-generation sequencing based method used to quantify circulating DNA in cancer (ctDNA). The method was introduced in 2014 by Ash Alizadeh and Maximilian Diehn’s laboratories at Stanford, as a tool for measuring Cell-free tumor DNA which is released from dead tumor cells into the blood and thus may reflect the entire tumor genome. This method can be generalized for any cancer type that is known to have recurrent mutations. CAPP-Seq can detect one molecule of mutant DNA in 10,000 molecules of healthy DNA. The original method was further refined in 2016 for ultra sensitive detection through integration of multiple error suppression strategies, termed integrated Digital Error Suppression (iDES). The use of ctDNA in this technique should not be confused with circulating tumor cells (CTCs); these are two different entities.

Circulating free DNA (cfDNA) (also known as cell-free DNA) are degraded DNA fragments released to body fluids such as blood plasma, urine, cerebrospinal fluid, etc. Typical sizes of cfDNA fragments reflect chromatosome particles (~165bp), as well as multiples of nucleosomes, which protect DNA from digestion by apoptotic nucleases. The term cfDNA can be used to describe various forms of DNA freely circulating in body fluids, including circulating tumor DNA (ctDNA), cell-free mitochondrial DNA (ccf mtDNA), cell-free fetal DNA (cffDNA) and donor-derived cell-free DNA (dd-cfDNA). Elevated levels of cfDNA are observed in cancer, especially in advanced disease. There is evidence that cfDNA becomes increasingly frequent in circulation with the onset of age. cfDNA has been shown to be a useful biomarker for a multitude of ailments other than cancer and fetal medicine. This includes but is not limited to trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplantation, diabetes, and sickle cell disease. cfDNA is mostly a double-stranded extracellular molecule of DNA, consisting of small fragments (50 to 200 bp) and larger fragments (21 kb) and has been recognized as an accurate marker for the diagnosis of prostate cancer and breast cancer.

Clinical metagenomic next-generation sequencing (mNGS) is the comprehensive analysis of microbial and host genetic material in clinical samples from patients by next-generation sequencing. It uses the techniques of metagenomics to identify and characterize the genome of bacteria, fungi, parasites, and viruses without the need for a prior knowledge of a specific pathogen directly from clinical specimens. The capacity to detect all the potential pathogens in a sample makes metagenomic next generation sequencing a potent tool in the diagnosis of infectious disease especially when other more directed assays, such as PCR, fail. Its limitations include clinical utility, laboratory validity, sense and sensitivity, cost and regulatory considerations.

Urinary cell-free DNA (ucfDNA) refers to DNA fragments in urine released by urogenital and non-urogenital cells. Shed cells on urogenital tract release high- or low-molecular-weight DNA fragments via apoptosis and necrosis, while circulating cell-free DNA (cfDNA) that passes through glomerular pores contributes to low-molecular-weight DNA. Most of the ucfDNA is low-molecular-weight DNA in the size of 150-250 base pairs. The detection of ucfDNA composition allows the quantification of cfDNA, circulating tumour DNA, and cell-free fetal DNA components. Many commercial kits and devices have been developed for ucfDNA isolation, quantification, and quality assessment.

Personalized genomics is the human genetics-derived study of analyzing and interpreting individualized genetic information by genome sequencing to identify genetic variations compared to the library of known sequences. International genetics communities have spared no effort from the past and have gradually cooperated to prosecute research projects to determine DNA sequences of the human genome using DNA sequencing techniques. The methods that are the most commonly used are whole exome sequencing and whole genome sequencing. Both approaches are used to identify genetic variations. Genome sequencing became more cost-effective over time, and made it applicable in the medical field, allowing scientists to understand which genes are attributed to specific diseases.

References

  1. 1 2 3 Poste G (May 2001). "Molecular diagnostics: a powerful new component of the healthcare value chain". Expert Review of Molecular Diagnostics. 1 (1): 1–5. doi: 10.1586/14737159.1.1.1 . PMID   11901792.
  2. 1 2 3 4 5 6 7 8 9 Burtis CA, Ashwood ER, Bruns DE (2012). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier. ISBN   978-1-4557-5942-2.
  3. ↑ "About ITP". ITP. Retrieved 23 April 2021.
  4. ↑ Hamburg MA, Collins FS (July 2010). "The path to personalized medicine". The New England Journal of Medicine. 363 (4): 301–4. doi: 10.1056/NEJMp1006304 . PMID   20551152. S2CID   205106671.
  5. ↑ Grody WW, Nakamura RM, Strom CM, Kiechle FL (2010). Molecular Diagnostics: Techniques and Applications for the Clinical Laboratory. Boston MA: Academic Press Inc. ISBN   978-0-12-369428-7.
  6. ↑ Kan YW, Lee KY, Furbetta M, Angius A, Cao A (January 1980). "Polymorphism of DNA sequence in the beta-globin gene region. Application to prenatal diagnosis of beta 0 thalassemia in Sardinia". The New England Journal of Medicine. 302 (4): 185–8. doi:10.1056/NEJM198001243020401. PMID   6927915.
  7. ↑ Cohn DV, Elting JJ, Frick M, Elde R (June 1984). "Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat". Endocrinology. 114 (6): 1963–74. doi:10.1210/endo-114-6-1963. PMID   6233131.
  8. ↑ Persselin JE, Stevens RH (August 1985). "Anti-Fab antibodies in humans. Predominance of minor immunoglobulin G subclasses in rheumatoid arthritis". The Journal of Clinical Investigation. 76 (2): 723–30. doi:10.1172/JCI112027. PMC   423887 . PMID   3928684.
  9. ↑ Kaplan G, Gaudernack G (October 1982). "In vitro differentiation of human monocytes. Differences in monocyte phenotypes induced by cultivation on glass or on collagen". The Journal of Experimental Medicine. 156 (4): 1101–14. doi:10.1084/jem.156.4.1101. PMC   2186821 . PMID   6961188.
  10. ↑ Fausto N, Kaul KL (1999). "Presenting the Journal of Molecular Diagnostics". The Journal of Molecular Diagnostics. 1 (1): 1. doi:10.1016/S1525-1578(10)60601-0. PMC   1906886 .
  11. ↑ Atanasovska B, Bozhinovski G, Chakalova L, Kocheva S, Karanfilski O, Plaseska-Karanfiska D (December 2012). "Molecular Diagnostics of β-Thalassemia". Balkan Journal of Medical Genetics. 15 (Suppl): 61–5. doi:10.2478/v10034-012-0021-z. PMC   3776673 . PMID   24052746.
  12. ↑ Sharples A (23 March 2011). "Gene Patents in Europe Relatively Stable Despite Uncertainty in the U.S." Genetic Engineering and Biotechnology News. Retrieved 13 June 2013.
  13. ↑ Bravin J, Kendall B (13 June 2013). "Justices Strike Down Gene Patents". The Wall Street Journal. Retrieved 15 June 2013.
  14. ↑ Barnes R, Brady D (13 June 2013). "Supreme Court rules human genes may not be patented". The Washington Post. Retrieved 15 June 2013.
  15. 1 2 Gibbs JN (1 August 2008). "Regulatory pathways for molecular diagnosis. Detailing the various options available and what each requires". 24 (14). Genetic Engineering & Biotechnology News . Retrieved 4 September 2013.{{cite journal}}: Cite journal requires |journal= (help)
  16. ↑ Gibbs JN (1 April 2011). "Uncertainty persists with RUO products. FDA may be considering more restrictive approach with research use only assays". 31 (7). Genetic Engineering & Biotechnology News . Retrieved 4 September 2013.{{cite journal}}: Cite journal requires |journal= (help)
  17. ↑ Tomiello K (21 February 2007). "Regulatory compliance drives LIMS". Design World. Retrieved 7 November 2012.
  18. ↑ Mackinnon AC, Wang YL, Sahota A, Yeung CC, Weck KE (November 2012). "Certification in molecular pathology in the United States: an update from the Association for Molecular Pathology Training and Education Committee". The Journal of Molecular Diagnostics. 14 (6): 541–9. doi: 10.1016/j.jmoldx.2012.05.004 . PMID   22925695.
  19. ↑ Rao JR, Fleming CC, Moore JE (7 July 2006). Molecular Diagnostics: Current Technology and Applications (Horizon Bioscience). Horizon Bioscience. p. 97. ISBN   978-1-904933-19-9.
  20. ↑ van Ommen GJ, Breuning MH, Raap AK (June 1995). "FISH in genome research and molecular diagnostics". Current Opinion in Genetics & Development. 5 (3): 304–8. doi:10.1016/0959-437X(95)80043-3. PMID   7549423.
  21. ↑ Hammerling JA (2012). "A Review of Medical Errors in Laboratory Diagnostics and Where We Are Today". Laboratory Medicine. 43 (2): 41–44. doi: 10.1309/LM6ER9WJR1IHQAUY .
  22. 1 2 3 4 5 6 7 Khodakov D, Wang C, Zhang DY (October 2016). "Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches". Advanced Drug Delivery Reviews. 105 (Pt A): 3–19. doi: 10.1016/j.addr.2016.04.005 . PMID   27089811.
  23. ↑ Sherwood, James L.; Müller, Susanne; Orr, Maria C. M.; Ratcliffe, Marianne J.; Walker, Jill (2014). "Panel Based MALDI-TOF Tumour Profiling is a Sensitive Method for Detecting Mutations in Clinical non Small Cell Lung Cancer Tumour". PLOS ONE. 9 (6): e100566. Bibcode:2014PLoSO...9j0566S. doi: 10.1371/journal.pone.0100566 . PMC   4067351 . PMID   24956168.
  24. ↑ Walter G, Büssow K, Lueking A, Glökler J (June 2002). "High-throughput protein arrays: prospects for molecular diagnostics". Trends in Molecular Medicine. 8 (6): 250–3. doi:10.1016/S1471-4914(02)02352-3. PMID   12067604.
  25. ↑ Sherwood JL, Brown H, Rettino A, Schreieck A, Clark G, Claes B, Agrawal B, Chaston R, Kong BS, Choppa P, Nygren AO, Deras IL, Kohlmann A (1 September 2017). "Key differences between 13 KRAS mutation detection technologies and their relevance for clinical practice". ESMO Open. 2 (4): e000235. doi:10.1136/esmoopen-2017-000235. PMC   5623342 . PMID   29018576.
  26. ↑ "Advances in Prenatal Molecular Diagnostics Conference (Introduction)". HealthTech. 2013. Retrieved 28 September 2013.
  27. 1 2 3 4 "Molecular Diagnostics - National Cancer Institute". Cancer.gov. 28 January 2005. Archived from the original on 26 September 2006. Retrieved 26 September 2013.
  28. ↑ Desta Z, Zhao X, Shin JG, Flockhart DA (2002). "Clinical significance of the cytochrome P450 2C19 genetic polymorphism". Clinical Pharmacokinetics. 41 (12): 913–58. doi:10.2165/00003088-200241120-00002. PMID   12222994. S2CID   27616494.
  29. ↑ Eggermann T, Spengler S, Gogiel M, Begemann M, Elbracht M (June 2012). "Epigenetic and genetic diagnosis of Silver-Russell syndrome". Expert Review of Molecular Diagnostics. 12 (5): 459–71. doi:10.1586/erm.12.43. PMID   22702363. S2CID   3427029.
  30. ↑ Tong CY, Mallinson H (May 2002). "Moving to nucleic acid-based detection of genital Chlamydia trachomatis". Expert Review of Molecular Diagnostics. 2 (3): 257–66. doi:10.1586/14737159.2.3.257. PMID   12050864. S2CID   26518693.
  31. ↑ Deyde VM, Sampath R, Gubareva LV (January 2011). "RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses". Expert Review of Molecular Diagnostics. 11 (1): 41–52. doi:10.1586/erm.10.107. PMID   21171920. S2CID   207219269.
  32. ↑ Pai M, Kalantri S, Dheda K (May 2006). "New tools and emerging technologies for the diagnosis of tuberculosis: part I. Latent tuberculosis" (PDF). Expert Review of Molecular Diagnostics. 6 (3): 413–22. doi: 10.1586/14737159.6.3.413 . PMID   16706743.
  33. ↑ Burkardt HJ (January 2011). "Pandemic H1N1 2009 ('swine flu'): diagnostic and other challenges". Expert Review of Molecular Diagnostics. 11 (1): 35–40. doi:10.1586/erm.10.102. PMID   21171919. S2CID   894775.
  34. ↑ Habibzadeh P, Mofatteh M, Silawi M, Ghavami S, Faghihi MA (February 2021). "Molecular diagnostic assays for COVID-19: an overview". Critical Reviews in Clinical Laboratory Sciences. 58 (6): 385–398. doi:10.1080/10408363.2021.1884640. PMC   7898297 . PMID   33595397.
  35. ↑ Han ET (March 2013). "Loop-mediated isothermal amplification test for the molecular diagnosis of malaria". Expert Review of Molecular Diagnostics. 13 (2): 205–18. doi:10.1586/erm.12.144. PMID   23477559. S2CID   24649273.
  36. 1 2 Tang YW, Procop GW, Persing DH (November 1997). "Molecular diagnostics of infectious diseases". Clinical Chemistry. 43 (11): 2021–38. doi: 10.1093/clinchem/43.11.2021 . PMID   9365385.
  37. ↑ Chiu CY, Miller SA (June 2019). "Clinical metagenomics". Nature Reviews. Genetics. 20 (6): 341–355. doi:10.1038/s41576-019-0113-7. PMC   6858796 . PMID   30918369.
  38. ↑ van Lier MG, Wagner A, van Leerdam ME, Biermann K, Kuipers EJ, Steyerberg EW, Dubbink HJ, Dinjens WN (January 2010). "A review on the molecular diagnostics of Lynch syndrome: a central role for the pathology laboratory". Journal of Cellular and Molecular Medicine. 14 (1–2): 181–97. doi:10.1111/j.1582-4934.2009.00977.x. PMC   3837620 . PMID   19929944.
  39. ↑ Shrimpton AE (May 2002). "Molecular diagnosis of cystic fibrosis". Expert Review of Molecular Diagnostics. 2 (3): 240–56. doi:10.1586/14737159.2.3.240. PMID   12050863. S2CID   27351636.
  40. ↑ Andorno R (October 2004). "The right not to know: an autonomy based approach". Journal of Medical Ethics. 30 (5): 435–9, discussion 439–40. doi:10.1136/jme.2002.001578. PMC   1733927 . PMID   15467071.
  41. ↑ Harmon, Amy (24 February 2008) Insurance Fears Lead Many to Shun DNA Tests. New York Times
  42. ↑ Minamoto T, Ougolkov AV, Mai M (November 2002). "Detection of oncogenes in the diagnosis of cancers with active oncogenic signaling". Expert Review of Molecular Diagnostics. 2 (6): 565–75. doi:10.1586/14737159.2.6.565. PMID   12465453. S2CID   26732113.
  43. ↑ Brennan DJ, O'Connor DP, Rexhepaj E, Ponten F, Gallagher WM (September 2010). "Antibody-based proteomics: fast-tracking molecular diagnostics in oncology". Nature Reviews. Cancer. 10 (9): 605–17. doi:10.1038/nrc2902. PMID   20720569. S2CID   23540973.
  44. ↑ Ferracin M, Veronese A, Negrini M (April 2010). "Micromarkers: miRNAs in cancer diagnosis and prognosis". Expert Review of Molecular Diagnostics. 10 (3): 297–308. doi:10.1586/erm.10.11. PMID   20370587. S2CID   45371217.
  45. ↑ Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, Valtorta E, Schiavo R, Buscarino M, Siravegna G, Bencardino K, Cercek A, Chen CT, Veronese S, Zanon C, Sartore-Bianchi A, Gambacorta M, Gallicchio M, Vakiani E, Boscaro V, Medico E, Weiser M, Siena S, Di Nicolantonio F, Solit D, Bardelli A (June 2012). "Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer". Nature. 486 (7404): 532–6. Bibcode:2012Natur.486..532M. doi:10.1038/nature11156. PMC   3927413 . PMID   22722830.
  46. ↑ Emmadi R, Boonyaratanakornkit JB, Selvarangan R, Shyamala V, Zimmer BL, Williams L, Bryant B, Schutzbank T, Schoonmaker MM, Amos Wilson JA, Hall L, Pancholi P, Bernard K (November 2011). "Molecular methods and platforms for infectious diseases testing a review of FDA-approved and cleared assays". The Journal of Molecular Diagnostics. 13 (6): 583–604. doi:10.1016/j.jmoldx.2011.05.011. PMC   3194051 . PMID   21871973.
  47. ↑ FDA-approved next-generation sequencing system could expand clinical genomic testing: experts predict MiSeqDx platform will make genetic testing more affordable for smaller labs. AM. J. Med. Genet. A 164, X-XI.
  48. ↑ Angulo B, Conde E, Suárez-Gauthier A, Plaza C, Martínez R, Redondo P, Izquierdo E, Rubio-Viqueira B, Paz-Ares L, Hidalgo M, López-Ríos F (27 August 2012). "A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry". PLOS ONE. 7 (8): e43842. Bibcode:2012PLoSO...743842A. doi: 10.1371/journal.pone.0043842 . PMC   3428292 . PMID   22952784.
  49. ↑ Gonzalez de Castro D, Angulo B, Gomez B, Mair D, Martinez R, Suarez-Gauthier A, Shieh F, Velez M, Brophy VH, Lawrence HJ, Lopez-Rios F (July 2012). "A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens". British Journal of Cancer. 107 (2): 345–51. doi:10.1038/bjc.2012.259. PMC   3394984 . PMID   22713664.
  50. ↑ Marchant J, Mange A, Larrieux M, Costes V, Solassol J (July 2014). "Comparative evaluation of the new FDA approved THxID™-BRAF test with High Resolution Melting and Sanger sequencing". BMC Cancer. 14: 519. doi: 10.1186/1471-2407-14-519 . PMC   4223712 . PMID   25037456.
  51. ↑ Salazar R, Roepman P, Capella G, Moreno V, Simon I, Dreezen C, Lopez-Doriga A, Santos C, Marijnen C, Westerga J, Bruin S, Kerr D, Kuppen P, van de Velde C, Morreau H, Van Velthuysen L, Glas AM, Van't Veer LJ, Tollenaar R (January 2011). "Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer". Journal of Clinical Oncology. 29 (1): 17–24. doi: 10.1200/JCO.2010.30.1077 . hdl: 2445/198405 . PMID   21098318.
  52. ↑ Wittner BS, Sgroi DC, Ryan PD, Bruinsma TJ, Glas AM, Male A, Dahiya S, Habin K, Bernards R, Haber DA, Van't Veer LJ, Ramaswamy S (May 2008). "Analysis of the MammaPrint breast cancer assay in a predominantly postmenopausal cohort". Clinical Cancer Research. 14 (10): 2988–93. doi:10.1158/1078-0432.CCR-07-4723. PMC   3089800 . PMID   18483364.
  53. ↑ Lee CC, McMillin GA, Babic N, Melis R, Yeo KT (May 2011). "Evaluation of a CYP2C19 genotype panel on the GenMark eSensor® platform and the comparison to the Autogenomics Infiniti™ and Luminex CYP2C19 panels". Clinica Chimica Acta; International Journal of Clinical Chemistry. 412 (11–12): 1133–7. doi:10.1016/j.cca.2011.03.001. PMID   21385571.
  54. ↑ Pfundt R, Kwiatkowski K, Roter A, Shukla A, Thorland E, Hockett R, DuPont B, Fung ET, Chaubey A (February 2016). "Clinical performance of the CytoScan Dx Assay in diagnosing developmental delay/intellectual disability". Genetics in Medicine. 18 (2): 168–73. doi: 10.1038/gim.2015.51 . hdl: 2066/167658 . PMID   25880438.
  55. ↑ Hovelson DH, McDaniel AS, Cani AK, Johnson B, Rhodes K, Williams PD, et al. (April 2015). "Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors". Neoplasia. 17 (4): 385–99. doi:10.1016/j.neo.2015.03.004. PMC   4415141 . PMID   25925381.
  56. ↑ Imperiale TF, Ransohoff DF, Itzkowitz SH (July 2014). "Multitarget stool DNA testing for colorectal-cancer screening". The New England Journal of Medicine. 371 (2): 187–8. doi:10.1056/NEJMc1405215. PMID   25006736.
  57. ↑ Rakovitch E, Nofech-Mozes S, Hanna W, Baehner FL, Saskin R, Butler SM, Tuck A, Sengupta S, Elavathil L, Jani PA, Bonin M, Chang MC, Robertson SJ, Slodkowska E, Fong C, Anderson JM, Jamshidian F, Miller DP, Cherbavaz DB, Shak S, Paszat L (July 2015). "A population-based validation study of the DCIS Score predicting recurrence risk in individuals treated by breast-conserving surgery alone". Breast Cancer Research and Treatment. 152 (2): 389–98. doi:10.1007/s10549-015-3464-6. PMC   4491104 . PMID   26119102.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.