Applications of gel electrophoresis and flow cytometry methodologies biology essay


Gel cataphoresis and flow cytometry are among of the most of import tools of biotechnology for the appraisal of DNA and protein in the cell. These of import methodological analysiss have been used in molecular biological science research labs for several decennaries to measure the molecular weight and structural parametric quantities of these supermolecules. In the past several old ages, these molecular biological science engineerings have become progressively of import as diagnostic tools in infirmary based clinical research labs for the designation of diseases, to measure molecular mechanisms of pathogenesis and to do intervention determinations. This paper is a reappraisal of the rules of each of these engineerings and their usage in clinical and molecular medical specialty.

Flow Cytometry

Analytic flow cytometry ( AFC ) is a powerful tool for measuring cell composing based on the appraisal of optical belongingss of cells scanned at the rate of about 100 cells per second ( Boddy et al 2001 ; Givan 2001 ) . Among the advantages of this engineering are rapid showing and the capacity to do quantitative measurings of single cells ( Davey & A ; Kell 1996 ; Givan 2001 ) . The technique has been applied to surveies in medical microbiology to place specific strains of bacteriums associated with patient infections ( Boddy et al 2001 ) .

It can besides be used to measure the effects of antibiotics on microbic strains to observe sensitiveness and opposition parametric quantities quickly to help the design and execution of therapy protocols in patients with acute infections Davey & A ; Kell 1996 ) . The technique can be used to mensurate the Deoxyribonucleic acid content within a cell, protein content and the activity of specific proteins within a cell ( Roederer 2001 ) . Flow cytometry is a extremely sensitive technique that involves measurings of fluorescence that are used to place and quantify biochemical belongingss of single cells ( Shapiro 2003 ) .

The method involves the sensing of optical excitement parametric quantities of fluorescent investigations applied to specific cellular constituents ( Shapiro 2003 ) . Optical excitement forms are standardized by controlled flow utilizing hydrodynamic focussing ( Shapiro 2003 ) . The application of multi-beam analysis such as two beam, two channel sensing and tom photon excitement systems that can be used to observe two fluorescent signals at the same time and a high signal to resound ratio ( Zhong et al, 2005 ) . Other applications include the usage of labelled nanoparticles to aim specific cell constituents for appraisal ( Zhong et al 2005 ) . Analytic flow cytometry can be used to measure rhythm rhythm parametric quantities in spliting cells on an single footing ( Pozaroski & A ; Darzynkiewicz 2004 ) . This may affect DNA content appraisal utilizing a fluorescent dye such as propidium iodide ( Shapiro 2003 ) In add-on bivariate analysis affecting the coincident appraisal of DNA content and proteins involved in cell rhythm ordinance can be used to decode the inside informations of this procedure in normal cells and malignant cells ( Pozaroski & A ; Darzynkiewicz 2004 ) . Cell viability is another measuring that can be made utilizing flow cytometry ( Bertho et al 2000 ) . Furthermore, the technique can be used to know apart between cell decease mechanisms associated with programmed cell death from mortification ( Bertho et al 2000 ) .

Cell viability measurings have of import clinical applications in pathogenesis to help in the elucidation of the tissue-destroying effects of disease. For illustration, the atomization of Deoxyribonucleic acid that occurs during programmed cell death causes a alteration in the spread belongingss of the molecule ( Bertho et al 2000 ) . Likewise, alterations in membrane permeabilization associated with mortification can besides be detected utilizing the methods of flow cytometry ( Bertho et al 2000 ) .

This tool can be really effectual in finding the extent of disease every bit good as the curative effects of intervention protocols in patients ( Galanzha et al 2008 ) . Another of import application of flow cytometry to the survey of physiology involves its usage in the sensing of protein: protein interactions. By agencies of fluorescence resonance energy transportation ( FRET ) to observe the interactions of proteins that are each tagged with a different fluorescent marker ( He et al 2003 ; Oswald 2004 ) . This engineering can be used to observe homotypic and heterotypic protein interactions that may be of import in placing cell tracts of import in specific disease processes ( He et al 2003 ; Maecker & A ; Trotter 2006 ) .

Highly specialized applications of flow cytometry have been utilized to analyze specific diseases. Another application of flow cytometry to clinical medical specialty has involved the designation of patients with heparin-induced thrombopenia, by agencies of the extremely sensitive sensing of anti-heparin: P4 antibodies that onslaught and destroy blood thrombocytes ( Gobbi et al 2004 ) . One of the more recent applications of flow cytometry to molecular medical specialty involves its usage in the appraisal of cistron hushing mechanisms by little microRNAs ( Martinex-Ferrandis et al 2007 ) . Inappropriate cistron silencing has been linked to several of import disease mechanisms include human malignant disease patterned advance in several types of malignant neoplastic disease ( He et al 2003 ) . Furthermore the usage of little interfering RNAs represents a fresh curative attack to the intervention of human diseases associated with the production of mutant proteins that cause tissue devastation ( Martinex-Ferrandis et al 2007 ) .

Flow cytometry appraisals affecting the usage of fluorescently labeled short interfering RNAs ( siRNAs ) have been used in cell screening experiments to observe cellular responses to these RNAs with possible curative application ( Maeker & A ; Trotter, 2006 ) . This sensitive appraisal facilitates the designation of cells in which the siRNA has efficaciously produced a cistron hushing consequence ( Martinex-Ferrandis et al 2007 ; Novo & A ; Wood 2008 ) .

Gel cataphoresis

Gel cataphoresis is a method of dividing DNA and protein supermolecules based on differences in molecular weight ( Voytas 2001 ) .

The basic format of this engineering involves the usage of an electric field to accomplish the separation of negatively charged molecules as they diffuse a gel polymer ( Voytas 2001 ) . The rate of migration of the supermolecule through the gel is determined by the diameter of the pores or gaps within the polymerized gel matrix ( Voytas 2001 ) . Due to its unvarying negative charges provided by the phosphate groups that comprise the sugar phosphate anchor of DNA, the DNA will migrate towards the positively charged electrode when placed in an electric field.

Furthermore, the rate of migration of additive DNA through the pore matrix of the gel polymer, will be determined straight by the length of the molecule or the figure of base braces in the two-base hit stranded spiral since the molecule has a fixed diameter ( Voytas 2001 ) . The migration form of additive Deoxyribonucleic acid can be used to find the molecular weight of different molecules when compared to the migration rate of DNA criterions of known molecular weight, estimated as the figure of base braces ( Voytas 2001 ) . The gel polymers used in DNA cataphoresis are agarose, which is used to analyse DNAs of molecular weights runing from about 200-25, 999 base brace ( bits per second ) , and polyacrylamide, which is used to divide low molecular weight DNAs ranging in size from 1-1000 bits per second ( Voytas 2001 ) . There are many of import applications of DNA cataphoresis in clinical medical specialty ( VanHeukelum & A ; Bartema 2003 ) . Deoxyribonucleic acid mutants that are associated with specific diseases can be identified to supply an of import diagnostic tool ( VanHeukelum & A ; Bartema 2003 ) .

In add-on DNA appraisal of disease pathogens can supply of import information on the etiologic agents associated with infective disease ( Sellers et al 2007 ) . Some applications of this engineering involve the testing of Deoxyribonucleic acid from malignant neoplastic disease patients as a tool for measuring the type of malignant neoplastic disease and its forecast ( Tse et al 2006 ) . For illustration, different mutants are associated with different types of chest malignant neoplastic disease ( Tse et al 2006 ) .

Inherited signifiers of the disease are associated with nmutations in the BRCA-1 and BRCA-2 cistrons, which is of import in forecast, disease return and intervention determinations ( Zustin et al 2009 ) . In the country of infective disease, the designation of specific viruses associated with flu eruptions is assisted by the analysis of the viral DNA composing ( Sellers et al 2007 ) . The comet check is an application of DNA gel cataphoresis that allows the designation of DNA harm in a individual cell ( Shapohnikov, 2008 ) . The cells to be assayed are lysed with detergent and embedded in an agarose gel. These embedded cells called nucleoids, migrate to the anode in a comet molded array whose tail strength is relative to the figure of dual strand interruptions in the cellular DNA ( Shaposhnikov 2008 ) . Protein gel cataphoresis besides requires a gel polymer and an electric field to divide proteins with different physical and biochemical belongingss ( Carrrette et al 2006 ) . The most common type of protein gel cataphoresis involves the usage of denaturing gels, in which a protein is assessed in its denaturized or unfolded constellation ( Daszykowski et al 2009 ) .

Protein denaturation is achieved with the usage of detergents and chemicals that break the chemical bonds that hold a protein in its native three- dimensional constellation ( Daszykowski et al 2009 ) . The denaturized protein is so coated with a dissolver, normally sodium dodecyl sulphate ( SDS ) which provides a unvarying negative charged on the natured protein to ease separation of different protein molecules based on differences in molecular weight ( Daszykowski et al 2009 ) . This method is used to find the molecular weight of proteins and can be used to compare the same protein in normal and diseases provinces ( Kaczmarek et al 2004 ) .

Protein gel cataphoresis can besides be carried out on native proteins in their 3D constellation to measure migration form difference based on amino acid permutations and unnatural conformational belongingss ( Carrette et al 2006 ) . New developments in gel cataphoresis engineering have resulted in more sophisticated disease appraisal tools in the infirmary research lab. Difference gel cataphoresis ( DIGE ) is a specialised application of standard gel cataphoresis that corrects for the intrinsic fluctuations in biomolecule cataphoresis migration forms produced by standard methods of gel cataphoresis ( Unlu et al 1997 ; Minden et Al 2009 ) . Standard methods of 2D protein cataphoresis in polyacrylamide gels involve the separation of 100s of cellular proteins at the same time in a individual gel which produces ineluctable perturbations in cataphoretic mobility of single proteins ( Dowsey et al 2008 ) . This built-in fluctuation in protein migration form makes it really hard to measure faithfully the consequences of different gel forms. This type of comparative appraisal is indispensable in clinical infirmary analyses of proteins associated with disease provinces in order to measure the physiological abnormalcies of protein construction and map that may play an of import function in disease procedures ( Dowsey et al 2008 ) .

This job was resolved by the innovation of a engineering that permitted the coincident appraisal of proteins from multiple cellular beginnings on the same gel ( Minden et al 2009 ) . This is accomplished by the usage of fiting sets of fluorescent dyes that facilitates a direct comparing of proteins from different beginnings ( Minden et al 2009 ) . In this manner, for illustration, proteins from malignant neoplastic disease cells can be straight compared with the same protein from a normal cell to measure whether alterations in the molecular weight or amino acerb sequence may correlate with the disease province of the tumour tissue ( Minden et al 2009 ) . Extra alterations that have added to the dependability of this process are the inclusion of pooled internal protein criterion controls and the usage of fractionated cell samples as the get downing stuff for protein extraction prior to cataphoretic separation ( Daszykowski et al 2009 ) . Discontinuous native protein gel cataphoresis is a method developed to measure the 3D structural constellation of a protein by gel cataphoresis ( Niepmann & A ; Zhang 2006 ) .

This method represents an application of standard non-denaturing protein gels that assess the migratory behaviour of proteins through polyacrylamide gels ( Niepmann & A ; Zhang 2006 ) . Standard gels of his type frequently can non be used to measure oligomeric proteins incorporating multiple fractional monetary units such as haemoglobin which is an alpha2-beta2 tetramer. The discontinuous method of protein gel cataphoresis permits the separation and declaration of proteins based on differences in molecular weight, form conformation and oligomeric province ( Rosell et al 2009 ) . This is accomplished by the usage of Serva bluish G to add negative charge to the proteins utilizing a discontinuous buffer and a gradient gel ( Raymer & A ; Smith 2007 ) . The amino acerb histidine is used alternatively of glycine in the gel running buffer to decelerate the migration rate of basic proteins ( Raymer & A ; Smith 2007 ) .

The gradient gel facilitates the differential separation of proteins based on conformation ( Sellers et al 2007 ; Tse et Al 2009 ) . Taken together, these alterations facilitate the structural appraisal of proteins from patients whose diseases are the consequences of protein abnormalcies, such as haemoglobin upsets ( ScarontastnAA? & A ; Scaronlais 2005 ) .


Flow cytometry and gel cataphoresis are standard engineerings used in molecular biological science research that have progressively been implemented in infirmary research labs in the survey of human disease. These engineerings provide sensitive tools for the analysis of DNA and protein supermolecules that play a function in the etiology of disease pathogenesis. There are many broad-spectrum applications of flow cytometry and gel cataphoresis in the clinical scene, runing from the designation of microbic pathogens, the designation of unnatural cellular proteins and the appraisal of faulty cistrons that may play an of import function in disease mechanisms. It is expected that the clinical applications of these engineerings to clinical medical specialty will go on to spread out and supply a greater apprehension of the molecular footing of disease, every bit good as the clinical showing and direction human diseases.