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Molecular assessment has increasingly become an essential diagnostic and assessment tool in clinical medicine. Among the most important of these tools of biotechnology are the technologies of gel electrophoresis and flow cytometry. Use of these molecular assessment tools has facilitated the study of DNA and proteins that may contribute to the pathogenetic mechanisms associated with diverse diseases. Mutations in DNA structure and abnormalities of protein structure and function can be identified using these molecular approaches. This paper is a review of the principles of each of these technologies as they are applied to clinical and molecular medicine.
Analytical flow cytometry (AFC) is used to assess the biochemical composition of cells using the optical properties of cells as they are scanned individually at a rapid rate (approximately 100 cells per second) through an optical scanner (Boddy et al 2001; Givan 2001). This methodology permits quantitative measurements of molecular components within individual cells (Davey & Kell 1996; Givan 2001). An important medical use of AFC is the identification of specific strains of infectious bacteria in infected cells obtained from patient biopsy (Boddy et al 2001). Once the pathogen is identified, AFC can also be used to assess the effects of therapeutics such as antibiotics on the clinical course of infection by examining patient cwells by AFC post-treatment. In this regard, AFC is an important biomedical tool in the assessment of parameters of clinical sensitivity and resistance of specific bacterial strains to specific therapeutic regimens (Davey & Kell, 1996). In addition, AFC can be used to measure the cellular DNA and protein content as well as the activity of specific enzymes (Roederer 2001).
The capability of AFC to assess molecular content within individual cells involves the use of fluorescence measurements to characterize the biochemical components of cells. In this technique, fluorescent probes are applied to specific cell molecules which are then assessed via the detection of optical excitation patterns emitted by these optically labeled cell components. (Shapiro 2003). These optical excitation patterns emitted by the fluorescently labeled cell components can be used in the identification of specific types of molecules and to quantitate their concentrations within the cell (Shapiro 2003). This accuracy is facilitated by the standardization of controlled flow by means of hydrodynamic focusing methods (Shapiro 2003). Further analytical sophistication can be achieved by the use of multi-beam analysis, involving two beam two channel detection parameters and photon excitation systems to detect two different cellular signals at the same time in the same scan while providing a high signal-to noise-ratio (Zhong et al 2005). Labeled nanoparticles can also be used as probes in the molecular assessment of cell composition (Zhong et al 2005).
In addition to biochemical composition, AFC can be used to assess cell proliferation by means of accurately identifying cell cycle composition of dividing mitotic cells. This assessment can be carried out in individual cells (Pozaroski & Darzynkiewicz 2004). This is commonly accomplished by the use of a DNA binding dye such as propidium iodide (Shapiro 2003). DNA and protein content can be assessed simultaneously in individual cells by means of bivariate analysis and comparisons between normal cells and tumor cells (Pozaroski & Darzynkiewicz 2004). This technique also permits the evaluation of cell viability on an individual basis and can distinguish between cell death mechanisms associated with apoptosis versus necrotic mechanisms (Bertho et al 2000). Apoptosis results in a characteristic cell fragmentation into small apoptotic bodies containing fragmented DNA segments; this process can be detected by AFC and distinguished from plasma membrane degradation which is characteristic of necrotic cell death mechanisms. Cell viability measurements are an important component of disease assessment with regard to the identification of pathogenic mechanisms that may cause cell death. Selective tissue destruction is an important clinical manifestation of infectious disease and can be monitored using AFC. Cell viability measurements by AFC therefore represent an important clinical evaluation tool for pathophysiological mechanisms that can also be used to assess the efficacy of therapeutic approaches designed to preserve cell viability (Galanzha et al 2008).
Flow cytometry can also be used to analyze protein:protein interactions using an application called fluorescence resonance energy transfer (FRET). In this approach, different proteins are labeled with different fluorescent tags that produce different excitation patterns based on the type of interaction (He et al, 2003; Oswald 2004). Homotypic and heterotypic protein interactions can be involved in pathological cellular responses important to disease mechanisms (He et al 2003; Maecker & Trotter 2006). AFC has also been used to assess the pathophysiology of specific diseases at the cellular level. The diagnosis of heparin-induced thrombocytopenia can be made using AFC to detect the presence of antibodies activated by heparin that selectively destroy blood platelets (Gobbi et al 2004). Another specific application of AFC is in Glanzmannââ‚¬â„¢s thrombasthenia. AFC can be used to diagnose this condition by identifying patient auto-antibodies involved in blood platelet destruction ( Giannini et al 2008). Yet another recently implemented molecular use of AFC involves the detection of gene silencing mechanisms resulting from the action of microRNAs. (Martinex-Ferrandis et al 2007). The inappropriate activity of these nucleic acid regulators has been linked to several types of cancer and other diseases (He et al 2003). MicroRNAs are also being studied for therapeutic applications to disable disease-causing proteins. AFC can be used to monitor and assess the activities of these important regulatory molecules (Martinex-Ferrandis et al 2007). Flow cytometry assessments involving the use of fluorescently labeled short interfering RNAs (siRNAs) have been used in cell sorting experiments to detect cellular responses to these RNAs with potential therapeutic application (Maeker & Trotter, 2006). Using this approach, the molecular basis of cell responses to miRNAs can be assessed at the molecular level (Martinex-Ferrandis et al 2007; Novo & Wood 2008).
Gel electrophoresis is a powerful tool for the assessment of DNA and protein molecules that can be used to identify these macromolecules based on their molecular weight (Voytas 2001). This technology involves the use of voltage to create an electric field to achieve the separation of negatively charged molecules as they migrate through the molecular matrix of a gel polymer, such as agarose or polyacrylamide (Voytas 2001). The rate of migration of linear DNA and denatured charged protein molecules through the gel is determined by the pore diameter within the polymerized gel matrix (Voytas 2001). The rate of migration of DNA is inversely proportional to the log of the molecular weight of the DNA. DNA carries a uniformly distributed net negative charge due to the phosphate groups that comprise the sugar phosphate backbone of the DNA double helix. Therefore, the DNA will migrate from the negative electrode to the positively charged electrode when placed in an electric field. The rate of migration of linear DNA through the gel is determined directly by the number of base pairs in the double stranded helix since the molecule has a fixed diameter (Voytas 2001). The distance migrated by an individual DNA segment on the gel is used to determine its molecular weight when compared to the migration rate of DNA standards of known molecular weight, measured as a function of the number of base pairs (bps) (Voytas 2001). Agarose is the most commonly used gel polymer used in DNA molecular weight determination, as the pore matrix generated by this gel polymer can be used to separate and identify DNAs of molecular weights ranging from approximately 200-25,999 base pairs (bps). Polyacrylamide is another gel polymer which is used to separate low molecular weight DNAs ranging in size from 1-1000 bps and is also used in DNA sequence analysis (Voytas 2001).
DNA electrophoresis is used extensively in clinical medicine (VanHeukelum & Bartema 2003). This biotechnology tool is used to assess DNA mutations that are associated with specific diseases (VanHeukelum & Bartema 2003). DNA analysis of infectious disease agents provides important information on disease pathogenesis as well as the identification of infectious agents (Sellers et al 2007). The recent, rapid identification of specific viruses associated with flu outbreaks was made possible by the analysis of the viral DNA composition (Sellers et al 2007). DNA gel electrophoresis is also used extensively in cancer screening and diagnosis (Tse et al 2006). Specific mutations associated with different types of breast cancer and many other types of cancer can be distinguished using gel electrophoresis (Tse et al 2006). In the case of breast cancer, inherited forms of this disease result from mutations in the BRCA-1 and BRCA-2 genes. The molecular assessment of these disease causing mutations is important in prognosis of disease recurrence and treatment decisions (Zustin et al 2009). The comet assay is a recent advancement in DNA gel electrophoresis technology that permits the identification of DNA damage in a single cell (Shapohnikov, 2008). In this method, detergent-lysed cells are embedded in an agarose gel. These embedded cells are called nucleoids. The nucleoids containing DNA migrate through the gel polymer to the anode in a comet shaped pattern. The intensity of the tail band is proportional to the number of double strand breaks in the cellular DNA (Shaposhnikov 2008).
Protein gel electrophoresis is another powerful application of gel electrophoresis and is based on similar methodology to DNA gel electrophoresis. In protein gel electrophoresis, a gel polymer placed in an electric field is used to separate proteins with different physical and biochemical properties (Carrrette et al 2006). Since proteins have a more complex and variable three-dimensional structure and non-uniform charge distribution such as occurs in linear DNA, proteins must be modified in several ways prior to electrophoresis in order to make a determination of molecular weight. This process is called denaturation and involves the chemical unfolding of globular proteins using detergent. A uniform negative charge distribution is achieved by coating the linearized protein with specialized detergents such as sodium dodecyl sulfate (SDS) which effects the distribution of sulfate groups along the length of the linearized molecule. The protein now resembles DNA in its uniform diameter and charge distribution, so that differences in molecular weight are directly proportional to the length of the protein molecules generated by the linkage of amino acids. (Daszykowski et al 2009). The molecular weight of an unknown protein is determined by comparing its rate of migration through a denaturing gel polymer composed of polyacrylamide and SDS with the migration rate of protein standards of known molecular weight, measured in daltons (d). Differences in the molecular weights of proteins in normal and diseased states may result from mutations that alter the genetic code specifying protein structure. These structural mutations may affect protein function and cause pathophysiological effects characteristic of many different disease states (Kaczmarek et al 2004). Protein gel electrophoresis can also be carried out on intact globular proteins, termed ââ‚¬Å“native proteinsââ‚¬Â to assess migration pattern difference based on amino acid substitutions and abnormal conformational properties (Carrette et al 2006). The electrophoresis of hemoglobin from patients with sickle cell disease was responsible for identifying the mutation in the B-globin gene responsible for this blood disorder.
Many new applications of protein gel electrophoresis have been developed in recent years that have greatly expanded its biomedical usefulness. Difference gel electrophoresis (DIGE) is one example of an advanced application of protein electrophoresis specialized application of standard gel electrophoresis that can be used to detect very subtle amino acid changes in proteins resulting from gene mutations (Unlu et al 1997; Minden et al 2009). Conventional protein gel assessment methodologies, which involve the simultaneous separation and analysis of many different cellular proteins, create gel migration distortions of protein electrophoretic patterns that decrease the sensitivity of the assay (Dowsey et al 2008). These pattern distortions complicate the assessment of single protein analysis so important in the molecular characterization of proteins implicated in human disease (Dowsey et al 2008). DIGE technology allows for the accurate assessment of many cells for an individual protein that may be present in different structural configurations depending on its source (Minden et al 2009). Fluorescent dyes are used to identify and compare proteins from different cells that are electrophoresed on the same gel (Minden et al 2009). An important clinical application of DIGE involves the assessment of proteins from cancer cells that can be directly compared with the same protein from a normal cell to assess to detect changes in protein structure and function that may be the result of cancer-causing mutations (Minden et al 2009). In many cases internal protein standard controls are included in DIGE assessment in order to increase the sensitivity of this biotechnology tool (Daszykowski et al 2009).
Another modification of protein gel electrophoresis with extensive clinical application is called discontinuous native protein gel electrophoresis. This methodology is used to analyze the three-dimensional structure of proteins (Niepmann & Zhang 2006). This represents an advance over non-denaturing protein gel technology in use for many decades that are restricted generally to the analysis of monomeric proteins comprised of a single strand of amino acids (Niepmann & Zhang 2006). Discontinuous protein electrophoresis involves the separation of non-denatured proteins based on differences in oligomeric state in addition to molecular weight and 3-D structure (Rosell et al 2009). This method involves the use of an agent called Serva blue G that is mixed with the protein to add negative charges to their native protein. The protein is placed in a gradient gel and a discontinuous buffer is used to achieve protein separation (Raymer & Smith 2007). The gel running buffer contains a nonstandard amino acid, histidine, in place of glycine, which results in a slower migration pattern associated with electrophoretic protein separation (Raymer & Smith 2007). In this format, proteins separation involves differences in diverse parameters including oligomeric configuration (Sellers et al 2007; Tse et al 2009). This technology has facilitated the assessment of complex proteins and protein:protein associations that are critical components of the molecular assessment of disease (ScarontastnÃÂ¡ & Scaronlais 2005).
The biotechnology tools, flow cytometry and gel electrophoresis, were developed to facilitate the molecular assessment of DNA and proteins to assist basic research in molecular biology. Over the past several decades, these important technologies have been adapted to the research of clinical medicine to afford a molecular analysis of human disease that is unprecedented in medical history. The study of individuals cells, genes and proteins have permitted biomedical researchers to develop an in-depth understanding of the causes of human diseases, the physiological parameters that distinguish healthy and disease states, and the basis for the development of novel preventive, diagnostic screening, and therapeutic approaches to infectious, metabolic and genetic disorders. The ongoing development of newer and more advanced methodologies of flow cytometry and gel electrophoresis suggest that their importance in clinical medicine and biomedical research will continue to afford new insights in the study of molecular medicine.