Description

A classification of various technologies used for molecular diagnostics is shown in Table 2.

Table 2. Molecular Diagnostic Technologies

Monoclonal antibody-based immunochemical tests. Antibodies are the most popular class of molecules providing molecular recognition needs for a wide range of applications. The development of monoclonal antibody-based immunochemical assays to measure antibodies and antigens has substantially contributed to the diagnostic assays used routinely in clinics today. Monoclonal antibodies also have an impact on diagnostic imaging technologies. Monoclonal antibody technology is limited, however, by the manner in which antigen-antibody interactions can be controlled and by the ability to consistently produce antibodies with appropriate affinity and specificity. Advances in recombinant antigen preparation and antibody engineering have been used to enhance the applications of this technology.

Southern blotting. The Southern blot is named after its inventor (38). DNA is cleaved by restriction endonucleases, and the fragments of DNA are then separated by size using gel electrophoresis. Next the DNA fragments are denatured to separate the 2 strands of DNA. The single-stranded DNA molecules are then blot transferred from the gel onto the surface of a solid-phase filter, which is an opposed nitrocellulose or nylon membrane. After the DNA is heat-fixed to the membrane, the specific DNA sequence can be identified by incubating the filter with a single strand of radioactive DNA (called the probe) complementary to the recombinant DNA sequence of interest. The resulting hybridized labeled DNA:DNA complex, which is bonded to the nitrocellulose or nylon framework, is developed on an x-ray film to identify the original DNA fragments.

DNA probes. A probe is a single-stranded segment of DNA that has the complementary DNA sequence to bind another segment of DNA that is also single-stranded. The specificity of a DNA probe resides in its nucleotide sequence. DNA probes are commercially available and are of 3 types: (1) cDNA derived from mRNA by using reverse transcriptase; this represents DNA sequences that are present only in exons; (2) genomic probes, which can be derived from any segment of DNA and may contain anonymous DNA sequences (ie, segments of DNA that are not genes and may not even have a known chromosomal location); (3) oligonucleotide probes that are synthesized according to the required DNA sequence. These are small (20 to 30 base pairs), which is a disadvantage if total genomic DNA is used because the small size of the probe produces nonspecific background hybridization. These are, however, effective if they are hybridized against amplified DNA.

In the past, radiolabeled probes were of limited use. With the availability of amplification methods, such as PCR, calorimetric methods are now being used more frequently. DNA probes are designed according to the infective organism or the defective gene to be located.

Pulsed-field gel electrophoresis. This technique enables separation of large DNA molecules in the size range of several megabase pairs. This is difficult to achieve with conventional gel electrophoresis. In pulsed-field electrophoresis, where 2 alternating fields are used, separation of DNA fragments depends on their conformational properties. DNA molecules change the native conformation and elongate in the direction of the electrical field. When the second field is applied at an angle to the first one, the DNA molecules relax and are forced to change their conformation before they start to migrate in the direction of the second field. The relaxation or the reorientation time is a physical constant and is related to the molecular weight of the DNA; large DNA molecules need more time to rearrange than smaller ones. This technique requires "rare cutting" restriction endonucleases that cleave DNA into much larger fragments than is possible with frequently used varieties. These large fragments can be cloned and used for DNA mapping.

The polymerase chain reaction. The polymerase chain reaction is a method of nucleic acid analysis for producing large amounts of a specific DNA fragment of a defined sequence and length from a small amount of a complex template. It can selectively amplify a single molecule of DNA or RNA several million-fold in a few hours. Use of this technology enables the detection and analysis of specific gene sequences in a patient's sample without cloning. Analyses can be performed on even a few cells from body fluids or in a drop of blood. Thus, PCR eliminates the need to prepare large amounts of DNA from tissue samples. Since its introduction a decade ago, PCR has revolutionized molecular diagnostics. In addition to laboratory diagnosis, PCR has also affected the fields of genomics and biotechnology.

Polymerase chain reaction is based on the enzymatic amplification of a fragment of DNA that is flanked by 2 "primers" or short oligonucleotides that hybridize to the opposite strands of the target sequence and then prime synthesis of the complementary DNA sequence by DNA polymerase (an enzyme). The chain reaction is a 3-step process of denaturation, annealing, and extension that is repeated in several cycles.

At each stage of the process, the number of copies doubles from 2, to 4, to 8, and so on. The reactions are controlled by changing the temperature; the first reaction is at 94°C, the second at 55°C, and the third at 72°C using a special heat-stable Taq polymerase. After 20 cycles, roughly 1 million copies exist, or enough material to detect the desired DNA by conventional means such as color reaction.

Polymerase chain reaction has been fully automated via the use of thermal cycling. It is a fast, sensitive, and specific test with applications in the diagnosis of various diseases.

Target selection. Several strategies are available for selecting a genetic target to be amplified to detect an infectious disease organism. For example, genes that contain both conserved and variable sequence regions may be targeted. In such a case, specificity may be obtained either at the amplification (primer) or detection (probe) stage. The target may also consist of a virulent gene that is uniquely responsible for distinguishing pathogenic from closely-related nonpathogenic strains, types, or species.

Detection of amplified DNA. The first detection methods used with PCR were radioactively labeled probes that identified specific amplified sequences. With improvements in specificity, it became possible to visualize amplified DNA of the predicted size directly by examining its fluorescence after staining. Probes have now been converted to nonisotopic colorimetric systems. In another approach, the probe is a "reverse" component (bound to a membrane) and "captures" a specific allele or a sequence variant if it is present in the amplified DNA.

Polymerase chain reaction has been the dominating basic technology in molecular diagnostics. One of the limitations of PCR is that it is not quantitative in its generic form because it is a sequence-dependent technique. Although the use of PCR in mutation analysis is well established in some well-defined human genetic lesions, an attempt is being made to extend these capabilities to detection of mutations in large genes or genes with multiple mutations that cover a wide range of sequence information. An effort is being made to combine PCR with sequence-independent methods.

Real-time quantitative PCR. This system includes an oligonucleotide probe composed of a 5' fluorescent reporter and a 3' quencher molecule. The probe is added to the PCR reaction, hybridizes downstream from an amplification primer, and is digested by the advancing polymerase, resulting in the release of the fluorescent reporter. The fluorescent signal can then be detected and recorded. Commercially available systems combine amplification with on-board detection and provide excellent quantitative results.

RNA diagnostics. Direct detection of RNA is desirable in some situations, particularly in the analysis of gene expression in human cells and tissues. Another important area of application is the detection of RNA viruses. The earliest test for this purpose was Northern blot. Reverse transcriptase PCR is the most sensitive technique for mRNA detection and quantitation currently available. RNA amplification enables direct detection of retroviruses such as HIV. Tests for RNA transcription include the following:

Reverse transcriptase PCR. This is more sensitive than Northern blots or coupled reverse transcription for measuring gene expression in tissues and cells. The technique consists of 2 parts: synthesis of cDNA from RNA by reverse transcription and amplification of a specific cDNA by PCR. It can be used for quantitation (ie, absolute amount of a specific sequence in a sample rather than simple detection of DNA by PCR). Several commercial methods are available for this purpose.

Cycling probe technology. This technique involves the introduction and multiplication of probes that are specific for the organisms being sought. Each probe is a sandwich of 2 short DNA segments attached to the 2 ends of an RNA segment. The probe attaches to a single strand of target DNA. Then the RNA strand is cut into 2 by RNase H, a naturally occurring enzyme. The 2 probe halves fall away and leave the target strand free for another probe to attach. After about 45 minutes, the probe fragments are collected and can be detected by a color change in the sample following a routine immunology-based procedure. In contrast to PCR, cycling probe technology is isothermal with the probes floating in a sample solution along with RNase at a constant temperature. Probe amplification is linear and not exponential, thus, eliminating carryover contamination, and gives a quantitative assessment of viral or bacterial load. Because a single cleavage step is involved, the test is easy, cheap to produce, and can be automated.

Nucleic acid sequence-based amplification. This offers a simple and rapid alternative method for nucleic acid amplification. Nucleic acid sequence-based amplification technology is based on simultaneous enzymatic activity of reverse transcriptase, RNA polymerase, and RNase in combination with 2 oligonucleotides. This technology depends on selective primer template recognition to drive a cyclical, exponential amplification of the target sequence. Unlike reverse transcriptase PCR, nucleic acid sequence-based amplification can selectively amplify RNA sequences in a DNA background because DNA strands are not melted out. There are no false positive signals due to dead bacteria. RNA targets permit the detection of live bacterial or viral activity following antibacterial or viral therapy.

Nonisotopic RNase cleavage assay. This method is based on RNase cleavage and can be used for the detection of mutations. To scan for mutations, targets are amplified by PCR using primers with 20-base phage promoters on their 5' ends. The crude PCR products are then transcribed in vitro, using phage RNA polymerase, and hybridized to form double-stranded RNA. Mutations in the test region result in base-pair mismatches when the complimentary reference and mutant transcripts are hybridized. After hybridization, the duplex RNA targets are treated with a mixture of RNAses capable of cleaving base pair mismatches on both strands. These are detected by staining under ultraviolet light. Applications of this test have been shown in detection of rifampin resistance in Mycobacterium tuberculosis, in loss of heterozygosity of the p53 tumor suppressor gene p53, and in detection of mutations in genetic disorders.

Alternatives to PCR: linked linear amplification of nucleic acids. This alternative to PCR is directed to the extensive amplification of a nucleic acid sequence of interest through a linked series of multi-cycle primer extension reactions. Each of the primers provides linear amplification of targeted DNA or RNA sequences. Linked linear amplification of nucleic acids has been shown to have similar performance to PCR in both target sequence yields and specificity. The necessary equipment for linked linear amplification is commonly found in the molecular pathology laboratory, and this procedure can easily be incorporated into existing amplification protocols. The composition of the assay primers greatly reduces the risk of sample contamination normally associated with DNA amplification using PCR.

Primer extension-dependent isothermal amplification technology. This is a novel and rapid nucleic acid probe-based technology to screen for infectious agents and genetic variants. Target nucleic acid extracted from the patient sample does not need to undergo PCR before it is probed because the bound probe is amplified in situ before detection and universal amplification, and detection reagents are used regardless of the target sequence. The only variables are the sample of target DNA and the probes themselves. Amplification takes place through the de novo synthesis of RNA from an RNA polymerase promoter sequence included in the probe, but this occurs only when both probes designed for each sequence are bound to their target. This unique action takes place at a 3-way junction between a DNA polymerase and an RNA polymerase. Advantages of primer extension-dependent isothermal amplification technology assay are that it can detect RNA or DNA targets, has target-specificity, and provides real-time quantitative detection.

Ligase chain reaction. Ligase chain reaction is a promising new technique often used in conjunction with primary PCR amplification. It employs a thermostable enzyme DNA ligase and allows the discrimination of DNA sequences differing in only a single base pair. The principle of ligase chain reaction is based in part on the ligation of 2 adjacent synthetic nucleotide primers that uniquely hybridize to 1 strand of the target DNA. Both ligated products can then serve as templates for the next reaction cycle, leading to an exponential amplification process analogous to PCR. If there is a mismatch at the primer junction, it will be discriminated against by thermostable ligase, and the primers will not be ligated. Therefore, the absence of ligated product indicates at least a single base-pair change to the target sequence. In an automated, nonradioactive format, ligase chain reaction enables detection of less than 10 molecules of DNA. Ligase chain reaction technique has an advantage over PCR in that it is faster because it requires 1 less incubation. Ligase chain reaction tests may be more sensitive than PCR because they amplify a much shorter region of the DNA. This would be particularly advantageous if the target DNA is damaged, which often occurs with the herpes simplex virus.

Molecular beacons. These are oligonucleotide probes that become fluorescent on hybridization. They consist of a stem-and-loop structure. The loop portion of the molecule is a probe sequence complementary to a predetermined target DNA, and annealing on either side of the probe sequence forms the stem. These can be used to monitor PCR product formation during or after the amplification process. These hairpin-shaped probes possess several advantages over linear fluorescent hybridization probes. Their use eliminates the need for gel analysis following thermal cycling, thereby saving time and decreasing the potential for contamination of laboratory equipment or reagents.

Target amplification technologies. Methods have been developed in which the signal, rather than the target, is amplified. These techniques include the following:

Branched DNA quantification test. The key feature of this test is a highly branched form of synthetic DNA reminiscent of a Christmas tree in shape. These novel molecules are produced by chemically adding long side chains to the normally linear DNA structure. The basic steps of the test are as follows:

Dendritic nucleic acid signal amplification. Dendritic molecules are highly-branched arborescent structures and can be extremely useful for the development of nucleic acid diagnostics as signal amplification tools. Furthermore, due to the relatively large size of nucleic acid molecules, nucleic acid dendrimers can be readily labeled with numerous fluorescent compounds and protein moieties. Dendritic DNA molecules can be readily labeled with numerous fluorescent compounds, enabling detection of single copy oligo-sequences in nanogram quantities of human DNA in Southern blot assay. Specificity to various DNA sequences is conferred to the dendrimers by hybridizing and covalently cross-linking oligonucleotides to the single-stranded surface of the dendrimers.

Q beta replicase system. In this system, the enzyme Q beta replicase amplifies the signal of the probe rather than the target nucleic acid. The probe is composed of an RNA molecule known as midvariant-1, and a specific RNA fragment corresponding to the organism can be detected. The probe RNA binds to the complementary target, and the Q beta replicase then replicates the RNA probe. This system can be used to detect nucleic acid sequences of between 500 and 1000 molecules. With this technique quantitating HIV-1 mRNA as targets, the limit of quantification is about 10,000 mRNA molecules.

Single-strand conformation polymorphism. Single-strand conformation polymorphism is based on the fact that the extent of migration of single-stranded DNA molecules through gel is determined by the molecules nucleotide composition. Thus, 2 strands of DNA that differ by a single base pair may be distinguished by use of single-strand conformation polymorphisms. This method is PCR-based and works best over DNA intervals of 200 base pairs or less. The method also requires some knowledge of the genomic organization of the transcript under investigation.

DNA sequencing. The genome sequence is an organisms blueprint: the set of instructions dictating its biological traits. The term DNA sequencing refers to methods for determining the exact order of the 3 billion nucleotide basesadenine, guanine, cytosine, and thyminethat make up the DNA of the 23 pairs of human chromosomes. Several techniques and machines are available for sequencing (Jain 2021d). Much remains to be accomplished to enable complete genome sequencing as a standard component of a patient's medical care. Currently available sequencing technologies are already improving molecular diagnostics. An example is that of Wilson disease where measurement of hepatic copper concentration has been the gold standard for diagnosis. Direct sequencing of the ATP7B gene is sensitive and specific and can obviate the need for invasive liver biopsy (04). A next generation sequencing system has been used to validate a panel for genetic diagnosis of over 100 inherited diseases including neurologic conditions, congenital hearing loss, and eye disorders (44). This study successfully demonstrated the feasibility of using this platform to carry out multiplex genetic tests for several rare diseases along with cloud computing for bioinformatics analysis as a relatively low-cost solution for implementation in clinical laboratories. The value of next generation sequencing in clinical neurology was demonstrated in a pilot study on 50 highly heterogeneous patients with ataxia where diagnosis could not be established despite extensive investigations (31). Use of bioinformatic tools to analyze the data predicted several different mutations in different genes of which some were novel, including 1 causing a newly described recessive ataxia syndrome. Next-generation sequencing technologies are opening new applications in healthcare. The most important areas of application are in diagnosis of cancer, genetic disorders, and infections. Because of the decreasing costs and relatively rapid time to results, next-generation sequencing-based testing is quickly becoming a standard of care for patients with rare neurogenetic diseases (41). In one third of patients with neurologic disorders of undetermined etiology, a definitive diagnosis can be reached with next-generation sequencing (28). Sequencing will contribute to the development of personalized medicine.

Next generation sequencing data have been applied to a cohort of children with severe specific language impairment to identify a noncoding specific language impairment-associated variant affecting gene regulation in the human brain as well as the affected gene (ARHGEF39), which represent new risk factors for specific language impairment (07). These findings show the importance of investigating regulatory variants when determining risk factors contributing to neurodevelopmental disorders.

Targeted whole-exome sequencing is a powerful diagnostic tool for a broad spectrum of heterogeneous neurologic disorders. As the costs are coming down and clinical benefits are likely to improve, whole-exome sequencing should be used early in the diagnostic workup of patients with unexplained early-onset epilepsy (06).

Although next-generation sequencing technologies enable rapid and inexpensive large-scale genomic analysis, thus, creating opportunities to integrate genomic data into the clinical diagnosis and management of neurologic disorders, the scale and complexity of these data make them difficult to interpret for use in neurology practice (Rexach et al 2019). A multidisciplinary approach that incorporates bioinformatics, clinical evaluation, and human genetics may be required to meet these challenges. Moreover, emergence of personalized neurology will facilitate application of genomic data analysis to patient-targeted therapies.

Cytogenetics. This study of chromosomes was carried out mostly by banding techniques that involve staining and identifying various regions on a chromosome. Although still in use, this method has been refined considerably by the application of molecular techniques such as fluorescent in situ hybridization, comparative genomic hybridization, and spectral karyotyping, and is called molecular cytogenetics. Cytogenetic abnormalities such as translocations, deletions, amplifications, inversions, and chromosome breaks often provide the first clue to the molecular basis of a disease (18).

Fluorescent in situ hybridization. In situ hybridization has emerged as a powerful and versatile tool for the detection and localization of nucleic acid sequences within an intact cell, chromosome, or tissue preparation by means of labeled complementary sequences. Development of in situ hybridization provides greater resolution than was possible via chromosomal banding. The essential steps of in situ hybridization procedure include preparation of the specimen, probe labeling, hybridization, and detection.

Fluorescent nucleotide labels are employed in fluorescent in situ hybridization techniques, including the highly effective nonisotopic fluorescent hybridization approach. Fluorescent in situ hybridization has 4 components: target, probes, fluorescent detection, and visualization. Several DNA probes, each labeled with a different fluorochrome, can be used in the same procedure so that separate loci can be identified and compared. Probes are hybridized to target DNA and tagged with a fluorochrome, either before or after hybridization. Numerous sources of these probes have been found, including oligonucleotides, PCR products, cDNA, and genomic fragments contained in plasmids. Several developments have taken place in fluorescent in situ hybridization technology during recent years. The most significant of these advances are as follows:

Direct visual in situ hybridization. A high-resolution, multicolored map can be obtained by stretching DNA in a linear fashion in conjunction with fluorescent hybridization techniques. This DNA mapping procedure, which is also known as direct visual in situ hybridization, offers a rapid alternative to a variety of standard mapping procedures. The extended DNA used in this method facilitates work at a fine resolution and provides good sensitivity with strings of signals from genomic fragment probes smaller than 5 kilobases.

Comparative genomic hybridization or "reverse painting." In this technique, the entire genomic DNA of a hybrid cell line is used as the probe. Consequently, all sequences present in the hybrid cell are identifiable on the target chromosomes. Comparative genomic hybridization is useful for detecting recurrent genomic alterations in solid tumors where chromosome banding is difficult to identify.

Automated fluorescent in situ hybridization. Automation is important for clinical application of fluorescent in situ hybridization. Probe amplification has, for example, been facilitated by the automation of PCR and in combination with the direct incorporation of labeled nucleotides. Fluorescent in situ hybridization with repetitive DNA probes has been quantified by digital image analysis. This method accelerates the fluorescent in situ hybridization procedure considerably and is suitable for quantitative microscopy. In addition, this technology has simplified fluorescent in situ hybridization techniques used to label chromosome regions with repetitive DNA probes, making this approach suitable for routine screening of numerical aberrations.

Fluorescent in situ hybridization with telomere specific probes. Telomeric regions, located at the ends of each chromosome, contain a high density of genes, thus, DNA probes for the terminal regions of the chromosome arms are proving to be useful tools for several disorders involving the telomeric regions. Deletions and rearrangements involving the tips of the chromosome arms have been described. Telomeric deletions and unbalanced translocations have been associated with idiopathic mental retardation and polycystic kidney disease. These telomeric regions represent a major diagnostic challenge in clinical cytogenetics because most of the terminal bands are G negative and have little banding resolution. Therefore, cryptic deletions and rearrangements can be impossible to detect by conventional methods.

Antisense oligonucleotides. These have been used in molecular probes for diagnosis. Antisense approaches have been used for diagnostic purposes as well, and some examples are as follows:

Peptide nucleic acid technology. Peptide nucleic acid is a synthetic DNA mimic, wherein the sugar backbone is replaced by a peptide backbone. This replacement allows for a molecule with peptide-like qualities in the areas of synthesis and modification, while retaining the ability to hybridize to complementary RNA or DNA with higher affinity and specificity than corresponding oligonucleotides. After the peptide nucleic acid probe is hybridized to a denatured DNA sample, gel electrophoresis separates the single-stranded DNA fragments by length. Detection of the bound peptide nucleic acid is possible with direct fluorescence by capillary electrophoresis. Labeled peptide nucleic acid can be used as a probe in fluorescent in situ hybridization, which has gained momentum as an important technology for the detection of infectious diseases and inherited and acquired genetic disorders. Early on, the unique properties of peptide nucleic acid were shown to confer superior performance over the corresponding DNA probes. The involvement of telomere shortening in cancer and aging is currently under investigation. Future modifications of these methods may enable analysis of telomere fluorescence in specific subpopulations of cells by including labeling of cells with monoclonal antibodies prior to hybridization. Peptide nucleic acid technology can be used for improving some shortcomings of PCR enabling quantification and for purification of the DNA used as the target for PCR. Peptide nucleic acid probes can be used as an alternative for Southern hybridization. Another new area of development is peptide nucleic acid-based biosensors.

Locked nucleic acids. Locked nucleic acids are a novel class of DNA analogues that provide tremendous improvements in a number of key properties of DNA. A locked nucleic acid combines the highest affinity ever reported for a DNA analogue for complementary DNA and RNA with a superb ability to discriminate between correct and incorrect target sequences. This property is extremely important for diagnostic uses. Another property that makes locked nucleic acids valuable for molecular diagnostics is that they are fully functional as part of either chimeric DNA/LNA, RNA/LNA, or fully modified LNA oligonucleotides that act as substrates for nucleic acid enzymes (eg, polymerases and kinases). This feature will facilitate significant simplifications of a range of current DNA diagnostic formats. No product based on this technology has yet reached the market.

MicroRNA (MiRNA)-based diagnostics. MiRNAs function at all stages of neuronal development, ranging from the initial specification of neuronal cell types to the formation and plasticity of synaptic connections between individual neurons. The abundant expression of many regulatory miRNAs in the human brain implies that their biological role should be tested by functional assays in neurons and by comparative expression profiling. Circulating miRNAs are found in several body fluids, including plasma or serum, CSF, urine, and saliva (23). There are significant differences between the circulating miRNA expression profiles of healthy individuals and those with disorders of the central nervous system. Circulating miRNA are potentially valuable biomarkers of neurodegenerative disorders, which can be measured by noninvasive techniques.

Links between miRNA dysfunction and neurologic diseases are being uncovered. Expression profiling of miRNAs in samples from various human cancers, including glioblastoma, has shown distinctive miRNA fingerprints, which can be used as diagnostic and prognostic tools.

Aptamers. The development of the systematic evolution of ligands by exponential enrichment process made possible the isolation of oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. These oligonucleotide sequences, referred to as "aptamers," are beginning to emerge as a class of molecules that rival antibodies in both therapeutic and diagnostic applications. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. Aptamers are nuclease-resistant and offer an alternative to antibodies as targeting and diagnostic agents. Other promising applications of aptamers are molecular imaging, cell imaging, and integration of imaging with therapy (37).

Proteomics-based diagnostics. A proteome is a set of functional proteins encoded by the genome, and the term proteomics indicates proteins expressed by a genome. Proteomics is the systematic analysis of protein profiles of tissues and parallels the related field of genomics. Proteome analysis is investigation of the expression pattern of all proteins in a tissue. Comparisons between normal and diseased tissues and the identification of changed protein expression levels will provide information for understanding disease processes. Some of these proteins may prove to be biomarkers of disease. Diagnostic technologies relevant to proteomics are as follows:

(1) 2-dimensional gel electrophoresis. The technique offers the highest resolution separations available for proteins when gels of adequate size are used. This level of resolution, almost 2 orders of magnitude better than any competing technique, makes 2-dimensional gel electrophoresis uniquely suited to the study of protein components of cells. By reference to the databases, individual proteins on the map can be identified as the product of genes that have been sequenced.

(2) 2-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The essence of this approach is to separate proteins from a specific cell or tissue type, record the pattern, and then produce a Western blot. Proteins in the blot are digested with a proteolytic enzyme that has well-defined cleavage specificity. Peptide fragments can be analyzed by matrix-assisted laser desorption mass spectroscopy. The resulting peptide masses are then compared with theoretical masses calculated from amino-acid sequence databases. Matrix-assisted laser desorption mass spectroscopy was 1 of the major breakthroughs in mass spectrometric methods for analysis of proteins. A modification of it, matrix-assisted laser desorption time of flight, involves the bombardment of a mixed matrix of solutes by laser pulse in an electric field. Velocity of distribution of absorbed ions has a broad range. Fast ions arrive in a lower mass but slow and fast ions arrive at the same time. When the masses of the fragments of proteins have been determined by mass spectrometry, they can be compared with the theoretical masses held in the databases in cases of organisms whose whole genomic sequences are known.

Amplifiable fluorescent probes for in vivo imaging. A unique class of optical probes with enzyme specificity is available for in vivo molecular imaging. This method is based on an optical imaging approach to visualize activated proteases in vivo using quenched near-infrared fluorescent probes that become detectable only after enzymatic activation. This method can be used as a gene therapy reporter. This approach has potential applications for tumor detection, tumor characterization, and in vivo evaluation of anticancer therapies. Optical in vivo imaging can be used to study brain function in stroke patients using intrinsic contrast to hemoglobin and for molecular imaging using external contrast.

Magnetic resonance imaging. MRI offers a noninvasive means to map brain structure and function by sampling the amount, flow, or environment of water protons in vivo. To permit a more direct imaging of the physiological state of cells or organs, several bifunctional contrast-enhancing agents for optical and magnetic resonance imaging of experimental animals have been synthesized, characterized, and tested in vivo. Employing this strategy allows the same biological structures of a specimen to be studied at dramatically different resolutions and depths. MRI contrast agents have been constructed in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage to increase the MR signal. The modulation is triggered by 3 types of biological events: enzymatic processing of the agent, binding of an intracellular messenger, and transfection of a selected plasmid. These agents represent the first examples of direct, 3-dimensional visualization of gene expression and intracellular second messenger concentration in the form of a 3-dimensional MRI.

Dextran-coated superparamagnetic iron oxide particles that are biocompatible can be internalized in cytoplasm and nuclear compartments of cells and demonstrated by fluorescence microscopy or immunohistochemistry. Labeled cells are highly magnetic and, thus, detectable by MRI. This method has potential applications for in vivo tracking of magnetically labeled cells by MRI and for recovering intracellularly labeled cells from organs. Potential applications are in tumor detection.

Magnetic resonance spectroscopy. MRS, a technique related to MRI, provides information about molecular structure of metabolites (36). It is useful in the diagnosis of both focal and diffuse central nervous system diseases. In some cases, it may provide similar or sometimes better diagnostic accuracy as compared to histopathology.

Molecular imaging. Molecular imaging is an emerging field of study that deals with the imaging of disease on a cellular and molecular level. It can be considered as an extension of molecular diagnostics. In contradistinction to "classical" diagnostic imaging, molecular imaging sets forth to probe the molecular abnormalities that are the basis of disease rather than to image the end effects of these molecular alterations. Several current in vitro assays for protein and gene expression have been translated into the radiologic sciences. MRI techniques are now capable of visualizing physiological and diseased processes at cellular and molecular levels, including cerebral blood flow, capillary perfusion and permeability, blood oxygenation level-dependent neuronal activation, and integrity of axonal fibers. Endeavors are under way to image targets ranging from DNA to entire phenotypes in vivo. The merging fields of molecular biology, molecular medicine, and imaging modalities may provide the means to screen active drugs in vivo, to image molecular processes, and to diagnose disease at a presymptomatic stage.

Molecular cell imaging can be carried out by the following techniques:

Epifluorescence microscopy. Cellular constituents such as proteins and nucleic acids can be observed using antibodies conjugated with fluorescent probes and illuminating the sample. This is a rapid method for obtaining high quality images of a sample, particularly if it is thin (eg, cell monolayers), but has a limited use if the specimen is more than 5 mm thick.

Confocal microscopy. This differs from conventional microscopy because it incorporates laser scanning and enables study of thick samples. It enables the observation of cells labeled with the marker green fluorescent protein.

Single molecule detection. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available currently, the number of DNA molecules of a sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule.

Biochip technology. Microarray or DNA chip technology (also called gene chip or "biochip") is a rapid method of sequencing and analyzing genes. It is comprised of DNA probes formatted on a microscale and the instruments needed to handle the samples (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatic tools). Hybridization of RNA- or DNA-derived samples on chips allows the monitoring of expression of mRNAs or the occurrence of polymorphisms in genomic DNA. Examples of application of biochip technology in molecular diagnostics are as follows:

Detection and identification of pathogenic bacteria. Multiple copies of bacterial DNA produced by PCR can be used to create "fingerprints" by electrophoresis in a silicon chip. These identifying marks can then be compared with "fingerprints" of known strains. Target DNA is extracted from the clinical sample and labeled with a fluorescent dye. Target sequences are then hybridized to the chip and paired with the corresponding probes on the chip, generating fluorescent signals at the site of the probe. Analysis of the resultant pattern of fluorescence enables identification of the target sequences and, thus, of the microbes present in the specimen.

Automated programmable electronic matrix. This microchip technology consists of a multisite, electronically controlled array of independent test areas, each capable of attracting, binding, or repelling DNA under specific conditions of charge, polarity, current, and voltage. The automated programmable electronic matrix microchip takes advantage of the well-established principles of electrophoresis by moving charged molecules in an electric field, but on a greatly miniaturized scale. As an example of this, DNA (which is strongly electronegative and, therefore, carries a net negative charge) can be moved in an electric field to an area of net positive charge. The sample DNA is significantly concentrated over time in the area of the positive charge. This concentrating effect facilitates and greatly speeds up the hybridization of DNA. This effect can simultaneously occur at each test site, permitting rapid, multiple tests on a single sample. Unwanted, nonspecific DNA is repelled from the area of the electrode under closely controlled electronic conditions.

Microfluidic devices. These are complete biochemical analysis systems that use nanoliter quantities of reagents and are referred to as labs-on-a-chip. Disposable, nonreusable chips are economical diagnostic devices.

Chromosome-on-a-chip. This technique is slightly different from a DNA chip in that it uses genomic DNA instead of cDNA. This technique has been found to be useful for tracking the chromosomal whereabouts of a gene. Further development of the technology will involve construction of a whole genome chip containing all the chromosomes on it and will be the equivalent of the present-day genetic linkage map.

Protein chip. This is comparable to DNA chip technology in the field of genome analysis and has important applications in the field of proteomics. The protein chip system uses small arrays or plates with chemically- or biologically-treated surfaces to interact with proteins. Unknown proteins are affinity captured on treated surfaces, desorbed and ionized by laser excitation, and detected according to molecular weight. Known proteins are analyzed using on-chip functional assays. For example, chip surfaces can contain enzymes, receptor proteins, or antibodies, enabling on-chip protein-to-protein interaction studies, ligand binding studies, or immunoassays. The system enables the detection and analysis of trace amounts of proteins directly from biological tissues and fluids, including proteins differentially expressed in disease (22).

Bioelectronic microchips. These chips contain numerous electronically active microelectrodes with specific DNA capture probes linked to the electrodes through molecular wires. Target DNA or RNA is labeled in this system by hybridization to specific signaling probes covalently labeled with ferrocene, a redox label. The microelectrode surface is electrically insulated with a monolayer coating to prevent unwanted redox species in the sample chamber from interfering with measurements. Signals, therefore, depend on specific probe and target interaction (ie, hybridization). Minimal specimen preparation is required, and the system works in whole blood and contaminated specimens. This technology detects, among other targets, single nucleotide polymorphisms and matches conventional DNA testing for genetic mutations.

Future potential of biochip technology. The potential of future applications of microarray technology is vast. It is likely to become as commonplace as PCR is now. It may be possible to combine the resolution and quantitative ability of DNA chips with the sensitivity and specificity of in situ PCR (22).

Biosensors. Biosensors incorporate a biological sensing element that converts a change in an immediate environment to signals that can be processed. Molecular biosensors are based on antibodies, enzymes, ion channels, or nucleic acids. In practice, however, development of nucleic acid sensor systems has been hampered by the challenges presented in sample preparation. Nucleic acid isolation remains the rate-limiting step for all state-of-the-art molecular analyses. Although the optical types of biosensors are the most commonly employed currently, the electrochemical ones are expected to give the lowest detection limits along with the simplest instrumentation for on-site and point-of-care applications.

Electrochemical detection of DNA. A method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood is designed for use at the point-of-care, particularly in resource-limited settings (40). By combining recombinase polymerase amplification, an isothermal alternative to the PCR, with an electroactive mediator, this electrochemical method enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer, which is specifically designed for this type of analysis.

Nanotechnology-based diagnostics. Nanotechnology extends the limits of molecular diagnostics to nanoscale, and nanotechnology on a chip is another dimension of microfluidic and lab-on-a-chip technology (17). Biological tests measuring the presence or activity of selected substances become quicker, more sensitive, and more flexible when certain nanoscale particles are put to work as tags or labels. Gold nanoparticles tagged with short segments of DNA can be used for detection of genetic sequence in a sample. Nanobarcodes, submicrometer metallic barcodes with striping patterns prepared by sequential electrochemical deposition of metal, show differential reflectivity of adjacent stripes enabling identification of the striping patterns by conventional light microscopy. This has applications in population diagnostics and in point-of-care hand-held devices.

Nucleic acid diagnostics has been dominated in the past by fluorescence-based assays that use complex and expensive enzyme-based target or signal amplification procedures. Many clinical diagnostic applications will require simpler, inexpensive assays that can be done in a screening mode. Nanotechnology-based diagnostics for point-of-care use are faster and less expensive than conventional laboratory-based molecular diagnostics.

Nanotechnology-based molecular imaging of the brain. An extension of molecular diagnostics is the use of superparamagnetic iron oxide nanoparticles as cell-specific contrast agents for imaging macrophages in stroke during MRI. Nanoparticle-induced signal alterations differ from signatures of conventional gadolinium-enhanced MRI and are, thus, independent from breakdown of the blood-brain barrier. An experimental MRI contrast agent using manganese oxide nanoparticles produces images of the anatomic structures of a mouse brain that are as clear as those obtained by histological examination. The new contrast agent will enable better diagnosis of neurologic disorders such as Alzheimer disease, Parkinson disease, and stroke.

Nanoparticles can cross the blood-brain barrier and improve the visibility of brain structures by MRI to aid in the early diagnosis of Alzheimer disease. One example is anti-Abeta immunomagnetic nanoparticles, which are biocompatible and biologically active, and could be used as potential MRI contrast agents and targeted carriers for early diagnosis and therapy of Alzheimer disease (43).

Application of nanobiotechnology to neuromolecular imaging combined with Nanobiosensors enables continuous video tracking of molecular neurotransmitters in both the normal physiologic and disease states with long-term operational capability (05). This technology can track a signal in real time with excellent temporal and spatial resolution directly from each patient's brain to a computer as subjects are behaving during movement, normal, and/or dysfunctional. This would be useful in the management of Parkinson disease.

Clinical applications

Clinical applications of molecular diagnostic technologies relevant to neurology are listed in Table 3.

Table 3. Clinical Applications of Molecular Diagnostic Technologies Relevant to Neurology

Biomarkers of neurologic disorders. A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, an alteration from normal at DNA, RNA, or protein level, or pharmacologic responses to a therapeutic intervention. The expression of a distinct gene can enable its identification in a tissue with none of the surrounding cells expressing the specific biomarker. Currently available molecular diagnostic technologies have been used to detect biomarkers of various diseases such as cancer, metabolic disorders, infections, and diseases of the central nervous system. Some of the newly discovered biomarkers also form the basis of innovative molecular diagnostic tests (16). Biomarkers relevant to neurologic disorders can be detected in blood or cerebrospinal fluid. Some biomarkers are also detected by brain imaging. Apart from their value for early diagnosis, they can form the basis for development of therapeutics for a disease and, thus, help to link diagnostics and therapeutics, which is an important feature of personalized medicine.

Discovery of biomarkers to diagnose and guide treatment of traumatic brain injury is problematic because of diversity of secondary effects that follow initial trauma. A systems biology approach to analyze the complex molecular pathways and networks that mediate the secondary cellular response to traumatic brain injury using bioinformatic tools that integrate such diverse data have been shown to facilitate identification of candidate biomarkers (08).

Body tissues and fluids for diagnostics. Molecular diagnostic procedures performed on a variety of body tissues and fluids such as blood and urine give a clue to neurologic disorders. Tissues of the nervous system and cerebrospinal fluid are the most relevant examinations. Neuropathologies involving loss of neurons and disturbances of neurotransmission may result in disease-specific alterations of neuronal and cerebrospinal fluid proteins, which are suitable for proteomic analysis. CNS proteomics may identify cell types and tissues contributing to the disease phenotype (20). Application of proteomic technologies to study the changes of cerebrospinal fluid peptides clarify the aberrant processing of large intact protein precursors, elucidate the molecular mechanisms of CNS disorders, and find biomarkers of disease. Proteomic analysis of brain tissue has been done as an extension of the study of cerebrospinal fluid proteins.

Applications in genetics. Polymerase chain reaction enables detection of genetic defects associated with inherited diseases and determination of genetic susceptibility to disease, as well as risk of passing disease to offspring in families with affected members.

Diagnosis of infections. Molecular diagnostics have had a considerable impact on diagnosis and management of infectious diseases. These tools have been developed in response to diagnostic methods that lack sensitivity, specificity, or rapid turnaround time. They also assist with identification of agents that are difficult to cultivate or classify, or as methods for assessing the effects of antiviral or antimicrobial agents in chronic infection. Molecular methods have also enabled microbiologists to define disease by the presence of virulence, toxin, or antimicrobial resistance genes and to identify potentially important clones of organisms responsible for outbreaks of infection.

Real-time PCR for COVID-19. A study has kept tabs on community transmission of the COVID-19 infection by screening samples from a large population, which includes asymptomatic/symptomatic individuals for detection of severe acute respiratory syndrome coronavirus 2 by real-time polymerase chain reaction (RT-PCR). Pooled-sample PCR analysis strategies can save substantial resources and time for COVID-19 mass testing in comparison with individual testing without compromising the resulting outcome of the test (10).

Patients with neurologic manifestations of COVID-19 were included in some studies, and the most common neurologic diseases were COVID-19-associated encephalopathy (30.2%), acute ischemic cerebrovascular syndrome (25.7%), encephalitis (9.5%), and Guillain-Barré syndrome; they appeared after the first COVID-19 symptoms with a median delay of 6 (3 to 8) days (29). Brain magnetic resonance imaging of encephalitis patients showed heterogeneous acute nonvascular lesions in 14 (66.7%) of 21. Cerebrospinal fluid was analyzed, with pleocytosis found in 18.6% and a positive SARS-CoV-2 PCR result in 2 patients with encephalitis.

Neuropathology and cancer. Analysis of samples of cells and tissues using molecular diagnostics and immunohistochemistry plays an important part in clinical pathology as a supplement to classical morphologic examination. DNA chip-based methods are used to screen for hundreds of mutations in a cancer gene and for rapid analysis of genetic changes within tumors of patients. DNA from tumors is cut up with restriction enzymes and labeled with fluorescent dyes. When placed in the DNA chip, the labeled sequences bind to their complement and fluoresce under an excitation light, identifying themselves to the reading computer by their position on the chip. Quantitative measurement of gene expression profiles of cancer cells in vivo offers a comprehensive view of the molecular anatomy of cancer. Molecular tumor analysis of hematological malignancies is now possible with advanced automated analysis using DNA arrays. This technique will likely influence brain tumor classification, which may still be based on morphologic aspects.

Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding from a biopsy specimen whether a brain tumor is a primary glial or an epithelial metastatic tumor in origin.

Pharmacogenomics. Molecular diagnostics has an important application in pharmacogenomics for examining the genetic basis for individual variations in response to drugs. Genetic polymorphisms are a major cause of individual differences in drug response. The traditional method of detection of these differences is by metabolic phenotyping, which is carried out by administering a drug and measuring the metabolites and clinical outcomes. Unfortunately, this approach tends to be labor intensive and requires repeated sample collections from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a certain trait. Commonly used genotyping methods include gel electrophoresis-based techniques (such as PCR coupled with restriction fragment-length polymorphism analysis), multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application has yet to be developed. Novel mutations or polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. Pharmacogenomics is an important basis for development of personalized or individually tailored medicines according to the genotype of a patient (14).

Monitoring of gene therapy. Molecular diagnostic procedures have several applications in gene therapy. Some of these are as follows:

Combination of molecular diagnostics with molecular therapeutics. PET has developed to the sophisticated stage of molecular imaging to provide a means for combination of molecular therapies and molecular diagnostics. It can be used for demonstrations of drug action at their sites of action in vivo and to assay biological outcomes of the processes being modified in the patient. This can facilitate the drug discovery and development process as well as improve the drug use in clinical practice. PET can detect biological changes early during the disease process, even in asymptomatic stages of neurodegenerative diseases several years before symptoms appear. Molecular techniques will not only facilitate the diagnosis of neurologic disorders before the clinical manifestations appear and apply preventive strategies if possible, but will also enable the development of personalized medicines (15; 14).