Molecular Diagnostics: Past, Present, And Future - Elsevier
Chapter 1Molecular Diagnostics: Past, Present,and FutureGeorge P. Patrinos1,2 and Wilhelm J. Ansorge31Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece;Erasmus University Medical Center, Faculty of Medicine and Health Sciences, Department of Bioinformatics, Rotterdam, The Netherlands;3Ecole Polytechnique Federal Lausanne, EPFL, Lausanne, Switzerland21.1 INTRODUCTIONMolecular or nucleic acid-based diagnosis of human disorders is referred to as the detection of the various pathogenic mutations in DNA and/or RNA samples in order tofacilitate detection, diagnosis, subclassification, prognosis,and monitoring response to therapy. Molecular diagnosticscombines laboratory medicine with the knowledge andtechnology of molecular genetics and has been enormouslyrevolutionized over the last decades, benefiting from thediscoveries in the field of molecular biology (see Table 1.1).The identification and fine characterization of the geneticbasis of the disease in question is vital for accurate provision of diagnosis. Gene discovery provides invaluableinsights into the mechanisms of disease, and gene-basedmarkers allow physicians not only to assess disease predisposition but also to design and implement improveddiagnostic methods. The latter is of great importance, asthe plethora and variety of molecular defects demands theuse of multiple rather than a single mutation detection platform. Molecular diagnostics is currently a clinical realitywith its roots deep into the basic study of gene expressionand function.1.2 HISTORY OF MOLECULARDIAGNOSTICS: INVENTING THE WHEELIn 1949, Pauling and his coworkers introduced the termmolecular disease into the medical vocabulary, based ontheir discovery that a single amino acid change at theβ-globin chain leads to sickle cell anemia, characterizedmainly by recurrent episodes of acute pain due to vesselocclusion. In principle, their findings have set the foundations of molecular diagnostics, although the big revolutionMolecular DiagnosticsCopyright 2010 Elsevier Ltd. All rights reserved.08 P374537 Ch01.indd 1occurred many years later. At that time, when molecularbiology was only hectically expanding, the provision ofmolecular diagnostic services was inconceivable and technically not feasible. The first seeds of molecular diagnostics were provided in the early days of recombinant DNAtechnology, with many scientists from various disciplinesworking in concert. cDNA cloning and sequencing were atthat time invaluable tools for providing the basic knowledge on the primary sequence of various genes. The latterprovided a number of DNA probes, allowing the analysisvia Southern blotting of genomic regions, leading to theconcept and application of restriction fragment length polymorphism (RFLP) to track a mutant allele from heterozygous parents to a high-risk pregnancy. In 1976, Kan andcoworkers carried out, for the first time, prenatal diagnosis of α-thalassemia, using hybridization on DNA isolatedfrom fetal fibroblasts. Also, Kan and Dozy, in 1978, implemented RFLP analysis to pinpoint sickle cell alleles ofAfrican descent. This breakthrough provided the means ofestablishing similar diagnostic approaches for the characterization of other genetic diseases, such as phenylketonurea (Woo et al., 1983), cystic fibrosis (Farrall et al., 1986),and so on.At that time, however, a significant technical bottleneckhad to be overcome. The identification of the disease causing mutation was possible only through the constructionof a genomic DNA library from the affected individual, inorder first to clone the mutated allele and then determineits nucleotide sequence. Again, many human globin genemutations were among the first to be identified throughsuch approaches (Busslinger et al., 1981; Treisman et al.,1983). In 1982, Orkin and his coworkers showed that anumber of sequence variations were linked to specificβ-globin gene mutations. These groups of RFLPs, termedhaplotypes (both intergenic and intragenic), have provided a18/18/2009 12:40:57 PM
2Molecular DiagnosticsTABLE 1.1 The timeline of the principal discoveries inthe field of molecular biology, which influenced thedevelopment of molecular diagnostics.DateDiscovery1949Characterization of sickle cell anemia as amolecular disease1953Discovery of the DNA double helix1958Isolation of DNA polymerases1960First hybridization techniques1969In situ hybridization1970Discovery of restriction enzymes and reversetranscriptase1975Southern blotting1977DNA sequencing1983First synthesis of oligonucleotides1985Restriction fragment length polymorphism analysis1985Invention of PCR1986Development of fluorescent in situ hybridization(FISH)1988Discovery of the thermostable DNA polymerase –Optimization of PCR1992Conception of real-time PCR1993Discovery of structure-specific endonucleases forcleavage assays1996First application of DNA microarrays2001First draft versions of the human genome sequence2001Application of protein profiling in human diseases2005Introduction of the high-throughput next-generationsequencing technologyfirst-screening approach in order to detect a disease-causingmutation. Although this approach enabled researchers topredict which β-globin gene would contain a mutation, significantly facilitating mutation screening, no one was in theposition to determine the exact nature of the disease-causingmutation, as many different β-globin gene mutations werelinked to a specific haplotype in different populations(further information is available at http://globin.bx.psu.edu/hbvar; Hardison et al., 2002; Patrinos et al., 2004;Giardine et al., 2007).At the same time, in order to provide a shortcut to DNAsequencing, a number of exploratory methods for pinpointing mutations in patients’ DNA were developed. The08 P374537 Ch01.indd 2first methods involved mismatch detection in DNA/DNA orRNA/DNA heteroduplexes (Myers et al., 1985a, b) or differentiation of mismatched DNA heteroduplexes using gelelectrophoresis, according to their melting profile (Myerset al., 1987). Using this laborious and time-consumingapproach, a number of mutations or polymorphic sequencevariations have been identified, which made possible thedesign of short synthetic oligonucleotides that were usedas allele-specific probes onto genomic Southern blots. Thisexperimental design was quickly implemented for thedetection of β-thalassemia mutations (Orkin et al., 1983;Pirastu et al., 1983).Despite the intense efforts from different laboratoriesworldwide, diagnosis of inherited diseases on the DNAlevel was still underdeveloped and therefore still not readyto be implemented in clinical laboratories for routine analysis of patients due to the complexities, costs, and timerequirements of the technology available. It was only aftera few years that molecular diagnosis entered its golden erawith the discovery of the most powerful molecular biologytool since cloning and sequencing, the polymerase chainreaction (PCR).1.3 THE PCR REVOLUTION: GETTINGMORE OUT OF LESSThe discovery of PCR (Saiki et al., 1985; Mullis andFaloona, 1987) and its quick optimization, using a thermostable Taq DNA polymerase from Thermus aquaticus(Saiki et al., 1988) has greatly facilitated and in principlerevolutionized molecular diagnostics. The most powerfulfeature of PCR is the large amount of copies of the targetsequence generated by its exponential amplification (seeFig. 1.1), which allows the identification of a known mutation within a single day, rather than months. Also, PCR hasmarkedly decreased or even diminished the need for radioactivity for routine molecular diagnosis. This has allowedmolecular diagnostics to enter the clinical laboratory for theprovision of genetic services, such as carrier or populationscreening for known mutations, prenatal diagnosis of inherited diseases, or in recent years, identification of unknownmutations, in close collaboration with research laboratories.Therefore, being moved to their proper environment, theclinical laboratory, molecular diagnostics could provide theservices for which they have been initially conceived.The discovery of PCR has also provided the foundationsfor the design and development of many mutation detectionschemes, based on amplified DNA. In general, PCR eitheris used for the generation of the DNA fragments to be analyzed, or is part of the detection method. The first attemptwas the use of restriction enzymes (Saiki et al., 1985) oroligonucleotide probes, immobilized onto membranes or insolution (Saiki et al., 1986) in order to detect the existinggenetic variation, in particular the sickle cell disease-causing8/18/2009 12:40:57 PM
Chapter 1 Molecular Diagnostics: Past, Present, and FutureFIGURE 1.1 The PCR principle. Thick and thin black lines correspondto the target sequence and genomic DNA, respectively; gray boxes correspond to the oligonucleotide primers, and the correct size PCR productsare included in the white ellipses. Dashed lined arrows depict the elongation of the template strand.mutation. In the following years, an even larger number ofmutation detection approaches have been developed andimplemented. These techniques can be divided roughly intothree categories, depending on the basis for discriminatingthe allelic variants:1. Enzymatic-based methods. RFLP analysis was historically the first widely used approach, exploiting thealterations in restriction enzyme sites, leading to thegain or loss of restriction events (Saiki et al., 1985).Subsequently, a number of enzymatic approaches formutation detection have been conceived, based on thedependence of a secondary structure on the primaryDNA sequence. These methods exploit the activity ofresolvase enzymes T4 endonuclease VII, and, morerecently, T7 endonuclease I to digest heteroduplexDNA formed by annealing wild-type and mutant DNA(Mashal et al., 1995). Digestion fragments indicate thepresence and the position of any mutations. A variationof the theme involves the use of chemical agents for thesame purpose (Saleeba et al., 1992; see also Chapter 3).Another enzymatic approach for mutation detection isthe oligonucleotide ligation assay (Landegren et al.,1988). In this technique, two oligonucleotides are08 P374537 Ch01.indd 33hybridized to complementary DNA stretches at sitesof possible mutations. The oligonucleotides’ primersare designed such that the 3 end of the first primer isimmediately adjacent to the 5 end of the second primer.Therefore, if the first primer matches completely withthe target DNA, then the primers can be ligated by DNAligase. On the other hand, if a mismatch occurs at the 3 end of the first primer, then no ligation products will beobtained.2. Electrophoretic-based techniques. This category ischaracterized by a plethora of different approachesdesigned for screening of known or unknown mutations, based on the different electrophoretic mobility ofthe mutant alleles, under denaturing or non-denaturingconditions. Single strand conformation polymorphism(SSCP) and heteroduplex (HDA) analyses (Oritaet al., 1989; see Chapter 4) were among the first methods designed to detect molecular defects in genomicloci. In combination with capillary electrophoresis (seeChapter 5), SSCP and HDA analysis now provide anexcellent, simple, and rapid mutation detection platformwith low operation costs and, most interestingly, thepotential of easily being automated, thus allowing forhigh-throughput analysis of patients’ DNA. Similarly,denaturing and temperature gradient gel electrophoresis(DGGE and TGGE, respectively) can be used equallywell for mutation detection (see Chapter 6). Inthis case, electrophoretic mobility differences betweena wild-type and mutant allele can be ‘‘visualized’’in a gradient of denaturing agents, such as urea andformamide, or of increasing temperature. Finally, anincreasingly used mutation detection technique isthe two-dimensional gene scanning, based on twodimensional electrophoretic separation of amplifiedDNA fragments, according to their size and base pairsequence. The latter involves DGGE, following the sizeseparation step.3. Solid phase-based techniques. This set of techniquesconsists of the basis for most of the present-day mutation detection technologies, since they have the extraadvantage of being easily automated and hence arehighly recommended for high-throughput mutationdetection or screening. A fast, accurate, and convenientmethod for the detection of known mutations is reversedot-blot, initially developed by Saiki and coworkers(1989) and implemented for the detection ofβ-thalassemia mutations. The essence of this methodis the utilization of oligonucleotides, bound to a membrane, as hybridization targets for amplified DNA.Some of this technique’s advantages is that one membrane strip can be used to detect many different knownmutations in a single individual (a one strip/one patienttype of assay), the potential of automation, and the easeof interpretation of the results, using a classical avidinbiotin system. However, this technique cannot be used8/18/2009 12:40:57 PM
4Molecular Diagnosticsfor the detection of unknown mutations. Continuousdevelopment has given rise to allele-specific hybridization of amplified DNA (PCR-ASO, Chapter 2) onfilters and recently extended on DNA oligonucleotidemicroarrays (see Chapters 16 and 17) for high throughput mutation analysis (Gemignani et al., 2002; Chanet al., 2004). In particular, oligonucleotides of knownsequence are immobilized onto appropriate surfacesand hybridization of the targets to the microarray isdetected, mostly using fluorescent dyes.The choice of the mutation detection method is dependent upon a number of variables, including the mutationspectrum of a given inherited disorder, the available infrastructure, and the number of tests performed in the diagnostic laboratory, and recently with issues of intellectualproperties (see also section 1.5.1 and Chapter 36). Mostof the clinical diagnostic laboratories have not investedin an expensive high technology infrastructure, since thetest volumes, that is, the number of tests expected to beperformed, have not been large enough to justify the capital outlay. Therefore, simple screening tests such as SSCPand HDA were and still are the methods of choice for manyclinical laboratories, as they allow for rapid and simultaneous detection of different sequence variations at a detection rate of close to 100%. Although PCR has significantlyfacilitated the expansion of molecular diagnostics, it nonetheless has a number of limitations. First of all, amplification of CG repeat-rich regions can be problematic for Taqpolymerase, which sometimes leads to the classic alternative of Southern blot analysis. Also, Taq polymerase is errorprone at a range of 10 4 to 10 5 per nucleotide, which isstrongly influenced by the conditions of the amplificationreaction, such as magnesium or deoxyribonucleotide concentration, pH, temperature, and so on. Polymerase errorscan contribute to unspecific background, depending on thedetection method, resulting in limiting the detection level.To overcome these technical problems, positive resultsshould be confirmed by alternative methods or by usinghigh fidelity thermostable polymerases.Finally, it needs to be stressed that despite the wealth ofmutation detection methodologies, DNA sequencing is stillconsidered the gold standard and the definitive experimental procedure for mutation detection. However, the costs forthe initial investment and the difficulties for standardizationand interpretation of ambiguous results have restricted itsuse only to basic research laboratories.1.4 MOLECULAR DIAGNOSTICS IN THEPOST-GENOMIC ERAIn February 2001, with the announcement of the firstdraft sequence of the human genome (International HumanGenome Sequencing Consortium, 2001; Venter et al., 2001)08 P374537 Ch01.indd 4and subsequently with the genomic sequence of other organisms, molecular biology has entered into a new era withunprecedented opportunities and challenges. These tremendous developments put pressure on a variety of disciplinesto intensify their research efforts to improve by orders ofmagnitude the existing methods for mutation detection, tomake available data sets with genomic variation and analyze these sets using specialized software, to standardizeand commercialize genetic tests for routine diagnosis, andto improve the existing technology in order to provide stateof-the-art automated devices for high-throughput geneticanalysis.The biggest challenge, following the publication of thehuman genome draft sequence, was to improve the existingmutation detection technologies to achieve robust cost-effective,rapid, and high-throughput analysis of genomic variation.In the last couple of years, technology has improved rapidlyand new mutation-detection techniques have become available, whereas old methodologies have evolved to fit intothe increasing demand for automated and high-throughputscreening. The chromatographic detection of polymorphic changes of disease-causing mutations using denaturing high-performance liquid chromatography (DHPLC; forreview, see Xiao and Oefner, 2001) is one of the new technologies that emerged. DHPLC reveals the presence of agenetic variation by the differential retention of homo- andheteroduplex DNA on reversed phase chromatography underpartial denaturation.Single-base substitutions, deletions, and insertions canbe detected successfully by UV or fluorescence monitoring within two to three minutes in unpurified PCR products as large as 1.5 kilo bases. These features, togetherwith its low cost, make DHPLC one of the most powerfultools for mutational analysis. Also, pyrosequencing, a nongel-based genotyping technology, provides a very reliablemethod and an attractive alternative to DHPLC (Chapter 8).Pyrosequencing detects de novo incorporation of nucleotides based on the specific template. The incorporationprocess releases a pyrophosphate, which is converted toATP and followed by luciferase stimulation. The lightproduced, detected by a charge coupled device camera,is ‘‘translated’’ to a pyrogram, from which the nucleotidesequence can be deducted (Ronaghi et al., 1998).The use of the PCR in molecular diagnostics is considered the gold standard for detecting nucleic acids and it hasbecome an essential tool in the research laboratory. Realtime PCR (Holland et al., 1991) has engendered wideracceptance of the PCR due to its improved rapidity, sensitivity, and reproducibility (see Chapter 7). The methodallows for the direct detection of the PCR product duringthe exponential phase of the reaction, therefore combining amplification and detection in one single step. Theincreased speed of real-time PCR is due largely to reducedcycles, removal of post-PCR detection procedures, and theuse of fluorogenic labels and sensitive methods of detecting8/18/2009 12:40:58 PM
Chapter 1 Molecular Diagnostics: Past, Present, and Futuretheir emissions. Therefore, real-time PCR is a very accurateand sensitive methodology with a variety of applications inmolecular diagnostics, allows a high throughput, and caneasily be automated and performed on very small volumes,which makes it the method of choice for many moderndiagnostic laboratories.Above all, the DNA microarray-based genotypingapproach offers simultaneous analysis of many polymorphisms and sequence alterations (see Chapters 16 and 17).Microarrays consist of hundreds of thousands of oligonucleotides attached on a solid surface in an ordered array.The DNA sample of interest is PCR amplified and thenhybridized to the microarray. Each oligonucleotide in thehigh-density array acts as an allele-specific probe andtherefore perfectly matched sequences hybridize more efficiently to their corresponding oligonucleotides on the array.The hybridization signals, obtained from allele-specificarrayed primer extension (AS-APEX) (Pastinen et al.,2000), are quantified by high-resolution fluorescent scanning and analyzed by computer software, resulting in theidentification of DNA sequence alterations. Therefore,using a high-density microarray makes possible the simultaneous detection of a great number of DNA alterations,hence facilitating genome-wide screening. Several arrayshave been generated to detect variants in the HIV genome(Kozal et al., 1996; Wen et al., 2000), human mitochondria mutations (Erdogan et al., 2001), β-thalassemia (Chanet al., 2004; Cremonesi et al., 2007), and glycose-6-phosphatedehydrogenase (G-6-PD) deficiency mutations (Gemignaniet al., 2002), and so on.In recent years, there has been a significant development of proteomics, which has the potential to becomean indispensable tool for molecular diagnostics. A usefulrepertoire of proteomic technologies is available, with thepotential to undergo significant technological improvements, which would be beneficial for increased sensitivity and throughput while reducing sample requirement(see Chapters 18 and 21). The improvement of these technologies is a significant advance toward the need for better disease diagnostics. The detection of disease-specificprotein profiles goes back to the use of two-dimensionalprotein gels (Hanash, 2000), when it was demonstratedthat leukemias could be classified into different subtypesbased on the different protein profile (Hanash et al., 2002).Nowadays, mass spectrometers are able to resolve manyprotein and peptide species in body fluids, being virtually set to revolutionize protein-based disease diagnostics(see Chapter 21). The robust and high-throughput natureof the mass spectrometric instrumentation is unparalleledand imminently suited for future clinical applications, aselegantly demonstrated by many retrospective studies incancer patients (reviewed in Petricoin et al., 2002). Also,high-throughput protein microarrays, constructed fromrecombinant, purified, and yet functional proteins, allowthe miniaturized and parallel analysis of large numbers08 P374537 Ch01.indd 55of diagnostic markers in complex samples. The first pilotstudies on disease tissues are already starting to emerge,such as assessing protein expression profiles in tissuederived from squamous cell carcinomas of the oral cavity(Knezevic et al., 2001), or the identification of proteinsthat induce an acute antibody response in autoimmune disorders, using auto-antigen arrays (Robinson et al., 2002).These findings indicate that proteomic pattern analysis ultimately might be applied as a screening tool for cancer inhigh-risk and general populations.The development of state-of-the-art mutation detection techniques has not only a positive impact on molecular genetic testing of inherited disorders, but also providesthe technical means to other disciplines. Mutation detectionschemes are applicable for the identification of geneticallymodified (GM) products, which may contaminate non-GMseeds, or food ingredients containing additives and flavoringsthat have been genetically modified or have been producedfrom GM organisms (see Chapter 29). The same techniquescan ascertain the genotype of an animal strain (see Chapter31). Another research area that benefits from the continuousdevelopment of mutation detection strategies is pharmacogenetics and pharmacogenomics (see Chapter 22), referred toas the effort to define the inter-individual variations that areexpected to become integral for treatment planning, in termsof efficacy and adverse effects of drugs. This approach usesthe technological expertise from high-throughput mutationdetection techniques, genomics, and functional genomics todefine and predict the nature of the response of an individualto a drug treatment, and to rationally design newer drugs orimprove existing ones. Ultimately, the identified genomicsequence variation is organized and stored into specializedmutation databases, enabling a physician or researcher toquery upon and retrieve information relevant to diagnosticissues (see Chapter 25).Finally, and for the last 20 years, DNA analysis andtesting has also significantly revolutionized the forensicsciences. The technical advances in molecular biologyand the increasing knowledge of the human genome havehad a major impact on forensic medicine (see Chapter 26).Genetic characterization of individuals at the DNA levelenables identity testing from a minimal amount of biological specimen, such as hair, blood, semen, bone, and soforth, in cases of sexual assault, homicide, and unknownhuman remains, and paternity testing is also changingfrom the level of gene products to the genomic level. DNAtesting is by far more advantageous over the conventionalforensic serology, and over the years has contributed to theacquittal of falsely accused people (saving most of themeven from death row) and the identification of the individual who had committed criminal acts (Cohen, 1995),and even helped to specify identities of unknown humanremains, such as those from the victims at Ground Zero inNew York, or from the skeletons of the Romanov familymembers (Gill et al., 1994).8/18/2009 12:40:58 PM
61.5 FUTURE PERSPECTIVES: WHAT LIESBEYONDAs an intrinsic part of DNA technology, molecular diagnostics are rooted in the April 1953 discovery of the DNAdouble helix. Today, it is clear that they embody a set ofnotable technological advances allowing for thousandsof diagnostic reactions to be performed at once and for arange of mutations to be simultaneously detected. The reasons for this dramatic increase are two-fold. First of all,the elucidation of the human genomic sequence, as well asthat of other species such as bacterial or viral pathogens,has led to an increased number of diagnostically relevanttargets. Second, the molecular diagnostic testing volumeis rapidly increasing. This is the consequence of a betterunderstanding of the basis of inherited diseases, thereforeallowing molecular diagnostics to play a key role in patientor disease management.Presently, a great number of blood, hair, semen, andtissue samples are analyzed annually worldwide in bothpublic and private laboratories, and the number of genetictests available is steadily increased year by year. Takingthese premises into account, we can presume that it is onlya matter of time before molecular diagnostic laboratoriesbecome indispensable in laboratory medicine. In the postgenomic era, genetic information will have to be examinedin multiple health care situations throughout people’s lives.Currently, newborns can be screened for phenylketonureaand other treatable genetic diseases (Yang et al., 2001). Itis also possible that in the not-so-distant future, childrenat high risk from coronary artery disease will be identifiedand treated to prevent changes in their vascular walls during adulthood. Similarly, parents will have the option ofbeing informed about their carrier status for many recessive diseases before they decide to start a family. Althoughnot widely accepted, this initiative has already started tobe implemented in Cyprus, where a couple at risk for thalassemia syndrome have been advised to undergo a genetictest for thalassemia mutations before their marriage (seealso Chapter 37). Also, for middle-aged and older populations, scientists will be able to determine risk profiles forvarious late-onset diseases, preferably before the appearance of symptoms, which at least could be partly preventedthrough dietary or pharmaceutical interventions. In the nearfuture, the monitoring of individual drug response profilesthroughout life, using genetic testing for the identification of their individual DNA signature, will be part of thestandard medical practice. Soon, genetic testing will comprise a wide spectrum of different analyses with a host ofconsequences for individuals and their families, which isworth emphasizing when explaining molecular diagnostics to the public (see also Chapter 38). All these issuesare discussed in detail next. However, and in order to bemore realistic, many of these expectations still are based onpromises, though quite optimistic ones. Thus, some of the08 P374537 Ch01.indd 6Molecular Diagnosticsnew perspectives of the field could be a decade away, andseveral challenges remain to be realized.1.5.1 Commercializing MolecularDiagnosticsCurrently, clinical molecular genetics is part of mainstreamhealth care worldwide. Almost all clinical laboratorieshave a molecular diagnostic unit or department. Althoughin recent years the notion of molecular diagnostics hasincreasingly gained interest, genetic tests are still not generally used for population screening, but rather for diagnosis,carrier screening, and prenatal diagnosis, and only on a limited basis. Therefore, and in order to make molecular diagnostics widely available, several obstacles and issues needto be taken into consideration and resolved in the comingyears.The first important issue is the choice of the mutationdetection platform. Despite the fact that there are over 50different mutation detection and screening methods, thereis no single platform or methodology that prevails forgenetic testing. Genotyping can be done using differentapproaches, such as filters, gels, microarrays, microtiterplates; different amplification-based technologies; differentseparation techniques, such as blotting, capillary electrophoresis, microarrays, mass spectroscopy; and finally different means for labeling, such as radioactive, fluorescent,chemiluminescent, or enzymatic substances. The varietyof detection approaches makes it not only difficult but alsochallenging to determine which one is better suited for alaboratory setting. Generally speaking, DNA sequencing is the gold standard for the identification of causativeor non-DNA sequence variations, particularly with theadvent of the next-generation sequencing technologies(see also Chapter 24). The initial investment costs and theexpected test volume are some of the factors that need tobe taken into consideration prior to choosing the detectiontechnique. Related issues are al
diagnostic methods. The latter is of great importance, as the plethora and variety of molecular defects demands the use of multiple rather than a single mutation detection plat-form. Molecular diagnostics is currently a clinical reality with its roots deep into the basic study of gene expression and function. 1.2 HISTORY OF MOLECULAR
IL PB BK DI8 DO4/EF-PAC 2-2 PHOENIX CONTACT 7725_en_01 2.1.1 Diagnostics in the IL PB BK DI8 DO4 format This diagnostic format consists of the following blocks: 1 PROFIBUS standard diagnostics 2 ID-specific diagnostics 3 Status diagnostics (terminal status) 4 Channel-specific diagnostics 5 Revision diagnostics (manufacturer-specific)
diseases, molecular diagnostics are among the most sen-sitive methods [1516, ]. Moreover, molecular diagnostics usually serve as the gold standard method for ongo-ing COVID-19 detection, allowing direct detection of virus-specic RNA or DNA regions prior to onset of the immune response [17, 18 ]. In the current review, we pre-
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