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On the Applicability of Electrophoresis for Protein Quantification

Karina dome.

1 Institute of Solid State Chemistry and Mechanochemistry, Siberian Branch, Russian Academy of Sciences, 630128 Novosibirsk, Russia; ur.liam@65aiozka (Z.A.); [email protected] (A.B.); ur.csn.dilos@vomol (O.L.)

Zoya Akimenko

Aleksey bychkov.

2 Department of Business, Novosibirsk State Technical University, 630073 Novosibirsk, Russia

Yuri Kalambet

3 Ampersand Ltd., 123182 Moscow, Russia; ur.dnasrepma@tebmalak

Oleg Lomovsky

Associated data.

The data presented in this study are available on request from the corresponding author.

Polyacrylamide gel electrophoresis is widely used for studying proteins and protein-containing objects. However, it is employed most frequently as a qualitative method rather than a quantitative one. This paper shows the feasibility of routine digital image acquisition and mathematical processing of electropherograms for protein quantification when using vertical gel electrophoresis and Chrom & Spec software. Both the well-studied model protein molecules (bovine serum albumin) and more complex real-world protein-based products (casein-containing isolate for sports nutrition), which were subjected to mechanical activation in a planetary ball mill to obtain samples characterized by different protein denaturation degrees, were used as study objects. Protein quantification in the mechanically activated samples was carried out. The degree of destruction of individual protein was shown to be higher compared to that of the protein-containing mixture after mechanical treatment for an identical amount of time. The methodological approach used in this study can serve as guidance for other researchers who would like to use electrophoresis for protein quantification both in individual form and in protein mixtures. The findings prove that photographic imaging of gels followed by mathematical data processing can be applied for analyzing the electrophoretic data as an affordable, convenient and quick tool.

1. Introduction

Protein chemistry methods are currently used to control, optimize, and elaborate novel technologies in molecular biology, pharmacology, bioengineering, and food technology [ 1 , 2 , 3 , 4 , 5 ]. Such efficient, fast, illustrative, and reproducible methods as high-performance liquid chromatography (HPLC) with different detectors and polymerase chain reaction (PCR) coupled with Sanger sequencing are used for protein quantification [ 6 , 7 , 8 , 9 ]. Despite the rapid progress in fast and efficient techniques employed for protein identification and quantification, simpler and more accessible analytical techniques (e.g., the conventional colorimetric measurements) also remain relevant [ 4 , 10 , 11 ]. Thus, these methods are used for Lowry protein assay in solutions in a reaction with the Folin reagent [ 11 ] or Bradford protein assay with Coomassie dye [ 12 ].

Protein-containing objects are usually analyzed by 1D and 2D polyacrylamide gel electrophoresis (PAGE), with sodium dodecyl sulfate (SDS) used as a detergent [ 10 , 13 , 14 , 15 , 16 , 17 ]. Staining with dyes that bind irreversibly to protein molecules but do not form stable bonds with polyacrylamide gel is often employed for protein detection in the gel [ 18 , 19 , 20 ]. The intensity of stained bands in gel depends on the amount of the applied sample; that is, it is assessed according to the laws of colorimetric measurements: staining intensity is directly proportional to protein content.

Electropherograms are illustrative and informative. However, this technique is most typically used as a qualitative method and quite rarely as a semi-quantitative test (only a visual assessment of band staining intensity is performed). The colorimetric approach (usually the visual one) is also employed in individual cases typically related to molecular biology for measuring the resolution during protein separation in polyacrylamide gel, as well as for protein quantification. In the quantification assay, electrophoretic separation is used together with enzyme-linked immunosorbent assay or western blotting [ 21 , 22 ], which requires respective immune sera against the target proteins.

In recent practical studies, there is demand for protein quantification in complex systems containing numerous impurities of protein and non-protein nature. Previously, polyacrylamide gel electrophoresis was used to determine the depth of hydrolysis of pea seed proteins [ 23 ]. The resulting hydrolysate enriched with free amino acids and peptides was used as a component of functional foods. The method combines the qualitative and quantitative assays of a protein mixture by polyacrylamide gel electrophoresis and simultaneous assessment of concentrations of the mixture components. It can also be used to develop special nutrition products containing pea seed proteins [ 24 ]. The topic of creating food products from peas is well developed, products containing peas have been mastered by the food industry and are popular [ 25 , 26 ]. In particular, this review notes the positive aspects of the technologies of dry processing of pea seeds.

Electrophoresis in polyacrylamide gel is no less popular in pharmaceuticals. Thus, in order to optimize the procedure for analyzing the drug aprotinin, the time-consuming chemical analysis was replaced by an analysis using HPLC [ 27 ]. Meanwhile, as aprotinin derivatives have a protein nature, they can be analyzed by polyacrylamide gel electrophoresis. The target aprotinin and its impurities can be detected by gel electrophoresis as clearly as by chromatography [ 28 ]. Thus, the methods of HPLC and electrophoresis in polyacrylamide gel can be interchanged. This approach can also be proposed for monitoring product purification in various bioengineering processes (novel forms of food products [ 24 , 29 ] or novel sorbents for protein purification [ 30 ]) and in the development of pharmaceuticals [ 31 ].

Therefore, this approach can be employed for manufacturing pharmaceutically important products, such as bovine serum albumin. As a result, simultaneous quantitative and qualitative monitoring of purification of the target product, albumin, will be useful in novel technologies [ 30 ].

The patent for an invention of a method for antibody isolation and purification can be mentioned as an example of using this technique for pharmaceutical products [ 31 ]. In this and similar studies, it is also convenient and efficient to perform manufacturing process monitoring and simultaneous quantitative assessment of concentrations of immunoglobulin components both during the purification stages and in the target products using PAGE.

The applicability of protein quantification by electrophoresis is currently limited by the following factors [ 32 ]. Firstly, there are certain difficulties related to obtaining digital images of the gels. The currently available scanners and densitometers are not common equipment; their resolution is insufficient to work with a densitogram like with a chromatogram. Secondly, the existing software mostly specializes in electrophoresis of nucleic acids and therefore uses a different signal-to-noise ratio [ 33 ].

This study makes a methodological attempt to use electrophoresis for protein quantification. The specially designed test bench for digital imaging of gels and optimally selected software allows one to quickly and easily determine the molecular weight distribution of protein molecules in the samples and perform a quantitative assay. This will enable quality control of protein products according to the quantitative contents of fractions of protein molecules and the presence of impurities. The software allows, if necessary, to calculate complex protein samples with diffuse (blurred) protein bands and to exclude unwanted, useless bands on the gel from the calculations. So, the processing of gels allows to obtain more information than other methods: the qualitative and quantitative composition of protein mixtures, as well as their molecular weight distribution.

2. Materials and Methods

Materials. Bovine serum albumin (BSA; #SLBB7759V, Sigma Aldrich, St. Louis, MO, USA) was used as the model study object. This protein was applied both in its non-modified form and after vigorous mechanical treatment. A protein-containing product, Kultlab Isolate ISO 90% sports nutrition supplement (Kultlab, Novosibirsk, Russia) with 90% casein content, was used as an experimental study object.

The following reagents were also used for electrophoresis analysis in polyacrylamide gel: acrylamide (Sigma Aldrich, St. Louis, MO, USA), SDS (Sigma Aldrich, St. Louis, MO, USA), N,N,N’,N’-tetramethylethylenediamine (Sigma Aldrich, St. Louis, MO, USA), glycine (Sigma Aldrich, St. Louis, MO, USA), tris base (Sigma Aldrich, St. Louis, MO, USA), ammonium persulfate (Sigma Aldrich, St. Louis, MO, USA), dithiothreitol (Sigma Aldrich, St. Louis, MO, USA), glycerol (Sigma Aldrich, St. Louis, MO, USA).

Mechanical treatment. In order to obtain samples characterized by various degrees of protein molecule destruction, BSA and the Kultlab sports nutrition supplement predominantly containing casein were subjected to mechanical treatment on an AGO-2 laboratory planetary ball mill (acceleration of the grinding media, 200 m/s 2 ). Treatment duration was varied between 5 and 30 min. The weight of the sample loaded into the reaction jars was 5 g per 200 g of the grinding media (steel balls 6 mm in diameter).

SDS-polyacrylamide gel electrophoresis was carried out using the Laemmli protocol [ 20 ]. Polyacrylamide (Sigma Aldrich, St. Louis, MO, USA) concentration in the stacking and resolving gels was 5% and 13%, respectively. The gel was stained using Coomassie R-250 dye (Thermo Fisher Scientific, Waltham, MA, USA). An unstained protein MW marker (Thermo Fisher Scientific, Waltham, MA, USA) with protein molecular weight ranging between 14.4 and 116 kDa was used as a protein marker.

BSA solution in a lysing buffer (2 mg/mL) for being applied onto the gel lanes was prepared according to the Laemmli protocol [ 20 ]. The calibration BSA solutions were prepared by twofold serial dilution. BSA concentration in the calibration solutions ranged from 0.0125 to 0.2 mg/mL. For calibration, the solutions were applied in such a manner that BSA concentration on the polyacrylamide gel lanes was sequentially reduced twofold. The samples of protein mixtures (components of sports nutrition) after mechanochemical activation were prepared using the same procedure. Dilution of sports nutrition samples was selected so that the band intensity lay within the calibration plot.

Protein quantification. In order to save the electrophoresis results, photos of the gel were taken with a camera (Olympus, Tokyo, Japan) with a 64 MP resolution. The photos were taken on a specially designed test bench with six light sources ensuring uniform illumination of the object ( Figure 1 ).

A schematic diagram of the photographic test bench: ( A ) top view; ( B ) side view.

Taking photos of the gel under these conditions allows one to obtain a densitogram with a resolution being manifold higher than the resolution attainable using scanners for gels. It will be demonstrated below that densitograms can be processed in the same way as chromatograms.

The grey-tone photo images of polyacrylamide gels with stained protein bands were used for protein quantification. Mathematical data processing was performed using the Chrom & Spec software in order to obtain a dependence between protein concentration and band color intensity/peak area [ 34 , 35 , 36 ]. The results of quantitative measurements were processed and saved using the Chrom & Spec software (Ampersend Ltd., Moscow, Russia).

3. Results and Discussion

As already mentioned, the electrophoresis results are most often assessed visually, and it is a qualitative assessment. In this study, the results were analyzed using the Chrom & Spec software consisting of two programs: the Planar software for image conversion to densitograms and the Multi Chrom-Planar software performing quantitative processing of densitograms ( Figure 2 ) [ 34 , 35 ]. The area of the resulting chromatographic peaks in the densitogram depends on band color intensity, which corresponds to protein content. The calculations were performed for each peak according to the standard operating procedure of the Chrom & Spec software. A more detailed description of the technical part of the work in the software can be found in Refs. [ 34 , 36 ].

Example of converting the electrophoretic profile of a protein marker into a densitogram using the Chrom & Spec software.

Figure 3 shows an example of the electropherogram of calibration BSA samples prepared by serial dilution. One can see that band staining intensity in the solutions applied onto the gel varies in accordance with protein content ( Figure 3 ).

( A ) An example of the electropherogram of calibration BSA solutions with concentrations 0.2, 0.1, 0.05, 0.025, and 0.0125 mg/mL, respectively (lanes 1–5) and the reference sample with known molecular weights (lane 6). Volume of the applied sample = 10 µL. ( B ) A densitogram of the reference sample.

The electropherogram was converted to densitograms using the Chrom & Spec software ( Figure 3 B). As a result of data processing, the area of analytical peaks depending on band color intensity in the gel was measured for the BSA samples with different protein concentrations (lanes 1–5). Table 1 summarizes the results of intensity measurements (peak area in arb. units).

The resulting data for plotting the calibration plot.

The resulting data were used to build a calibration plot “protein concentration vs. peak area” for BSA (the calibration protein) ( Figure 4 ). A quadratic calibration dependence was obtained:

where Q is the protein content (µg), and S is the area of the chromatographic peak on the densitogram. This dependence is standard for planar chromatography or gel electrophoresis [ 37 , 38 ]. The relative deviation was 3.8%. The molecular weight of the analyzed bovine serum albumin (68.53 kDa) was determined using the known molecular weights of the protein marker. The results correlate with the UniProt database values [ 38 , 39 ]. For further studies, the calibration and test samples were applied to the same gel. In this case, all the calculations conducted for the same gel prevent the problems related to the possible non-uniformity of background staining and differences in gel concentration.

The calibration plot for BSA quantification.

In order to obtain BSA samples characterized by different degradation degrees, they were subjected to mechanical treatment for different times. Sample concentration for electrophoresis analysis was selected so as the intensity of the stained bands lay within the calibration curve plotted for native BSA (shown in the same gel on lanes 6–10) ( Figure 5 ).

( A ) Electropherogram of the BSA degradation products after mechanical treatment for 5, 10, 15, 20, and 30 min (lanes 1–5) and the calibration BSA samples with concentration ranging from 0.0125 to 0.2 mg/mL (lanes 6–10); volume of the applied sample = 10 µL. ( B ) Densitograms of the BSA degradation products after mechanical treatment for 5 and 30 min (lanes 1 and 5).

The calibration plot was used as the standard of quantitative measurements to calculate the amount of BSA remaining in the sample after mechanical treatment ( Figure 6 ). Figure 6 shows the data on the degree of degradation (α) of protein molecules calculated using the formula:

where ∆ S is the change in the area of the peak corresponding to the native protein molecule after mechanical treatment (treatment duration t); S t is the area of the peak corresponding to the native protein molecule after mechanical treatment (treatment duration t); and S 0 is the area of the peak corresponding to the native protein molecule before mechanical treatment. Protein molecules in BSA subjected to 30-min mechanical treatment were degraded by 92 ± 3%.

Protein content in the samples of mechanically treated BSA.

The experiment involving mechanical treatment of milk protein isolate (a sports nutrition mix) and quantitative calculation of casein degradation products during this treatment was conducted in a similar way. Figure 7 shows the electropherogram of the samples of milk protein isolate before and after mechanical treatment for 5, 10, 15, 20, and 30 min. One can see that the destruction of casein protein molecules also takes place during mechanochemical treatment ( Figure 7 ).

( A ) Electropherogram of the initial sample of milk protein isolate (lane 1) and the samples of milk protein after mechanical treatment for 30, 20, 15, 10, and 5 min, respectively (lanes 2–6), as well as the calibration BSA samples with concentration ranging from 0.125 to 2.0 mg/mL (lanes 7–10). ( B ) Densitograms of BSA degradation products after mechanical treatment for 5 and 30 min (lanes 2 and 6).

The BSA calibration plot was used to obtain a dependence that allowed one to calculate casein content in the samples (in µg) before and after mechanical treatment for 5, 10, 15, 20, and 30 min. The results are shown in Figure 8 . It was demonstrated that the degree of degradation of protein molecules within the sports nutrition product after mechanical treatment for 30 min was 85 ± 2%, being comparable to the data for an individual protein (BSA). The diffusivity (polydispersity) of protein bands as a result of mechanical processing is also noted. This effect is observed in the case of casein proteins, as well as to a large extent for pea proteins in previous research [ 23 ]. This circumstance was an important factor for choosing the method of quantitative calculation of protein in the gel, namely, the choice of the Chrom & Spec software.

Protein content in milk protein isolate before and after mechanical treatment for 5, 10, 15, 20, and 30 min.

For the purpose of industrial use of the methodology of quantitative calculation of milk proteins in the gel, a Thermo Fisher Scientific kit has been developed and is used. It includes the following equipment [ 40 ]. The procedure allows the quantification of the protein in the gel for various technological tasks, such as the control of protein impurities in dairy products, the regulation of the content of target protein substances with simultaneous control of the distribution of molecular weight. However, for unique research work, such a kit may be inconvenient, expensive, and unavailable. An application for smartphones has been developed for the express processing of gels, which allows the determination of the molecular weights of fragments of deoxyribonucleic acid or proteins with high accuracy [ 41 ]. This approach is very interesting because its use does not require specific equipment. However, this application does not allow quantifying analytes. The method proposed in this article allows you to quickly obtain information about the molecular mass distribution of molecules in the gel, as well as the quantitative and qualitative composition. Also, the Chrom & Spec software allows the use of not only standard operating procedures but to choose the appropriate mathematical processing independently, depending on the task set by the researchers. This requires enough standard equipment such as a personal computer with the necessary software and a camera (it is also possible to use a scanner or smartphone), so this approach is available to a large number of researchers working with electrophoresis.

Hence, it has been shown that polyacrylamide gel electrophoresis coupled with simultaneous recording photographic images of the gels and mathematical data processing using the Chrom & Spec software allows one to measure protein content in the test sample directly in polyacrylamide gel (identically to the known colorimetric methods for protein quantification). This technique has made it possible to estimate the degree of protein degradation for the model BSA protein and casein (a component of sports nutrition products). The procedure allows protein quantification for various applied problems such as performing control over protein impurities or regulating the content of target protein substances with simultaneous control over the molecular weight distribution.

4. Conclusions

In this work, we used photographic visualization of gels followed by mathematical data processing using Chrom & Spec software to quantify the intensity of protein bands in polyacrylamide gel. The results obtained proved that this algorithm can be used to process electrophoretic data and obtain accurate data regarding the quantitative analysis of proteins using this method. The relative inaccuracy of the method was estimated using calibration solutions of BSA. To make the protein quantification more accurate, it was proposed to use calibration solutions together with test samples on a single gel so that all variable factors could be taken into account during the analysis and recording of the results.

The possibilities of the presented method were tested on BSA and casein in the composition of a sports nutrition product, which were subjected to mechanical processing. The proposed method was used to obtain data on the relationship between the degree of degradation of protein molecules and the duration of mechanical processing. Mechanical treatment of BSA for 30 min resulted in degradation of protein molecules by 92 ± 3%, while protein molecules in a sports nutrition product were degraded by 85 ± 2%. The degree of destruction of an individual protein was higher compared to the degree of destruction of a protein-containing mixture after mechanical treatment for an identical period of time.

The methodological approach used in this study can serve as a guide for other researchers who would like to use electrophoresis to quantify protein both in individual form and in protein mixtures.

Author Contributions

Conceptualization, A.B.; funding acquisition, A.B.; investigation, K.D. and Z.A.; methodology, Z.A., A.B. and Y.K.; software, Y.K.; resources, A.B. and O.L.; writing—original draft preparation, K.D. and Z.A.; writing—review and editing, A.B., Y.K. and O.L.; visualization, K.D. All authors have read and agreed to the published version of the manuscript.

The research was funded within the state assignment to ISSCM SB RAS (project No. FWUS-2021-0005). Determination of energy consumption was carried out with the support of the Russian Science Foundation (project No. 19-73-10074).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Data availability statement, conflicts of interest.

The authors declare no conflict of interest.

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Electrophoresis: Principles, Types, and Uses

Electrophoresis is a simple and sensitive separation technique in clinical and research laboratories. Since its discovery, it has been an essential tool used by biologists and chemists to separate mixtures, especially proteins and nucleic acids.

Electrophoresis consists of two words; electro , meaning electricity, and phoresis, meaning movement. Thus, it implies the migration and separation of a charged particle (ions) through a solution under the influence of an electric field.

It was first demonstrated in 1807 by Ruess, who noted the migration of particles towards the anode. It has improved from the initial crude paper electrophoresis to the modern automated system. Various improved versions are available, which apply in miniaturization, precision engineering, etc. The benefit of advancement is meeting the requirement for faster and better resolution of results.

Table of Contents

Principle of Electrophoresis

Biological molecules, like amino acids, peptides, proteins, nucleic acids, and nucleotides, possess ionizable groups. These molecules exist in solution as electrically charged species, cations (+), or anions (-) at any given pH. Thus, the electric field allows the migration of the negatively charged molecule towards the anode (a positive terminal). In contrast, the positively charged molecule migrates towards the cathode (a negative terminal).

The separation of the molecules, ions, or colloidal particles suspended in the matrix occurs due to the force of an electric field. The molecules move through a sieve-like compound based on the molecular mass and charge ratio.

It is an incomplete form of electrolysis as the electric field is removed before the molecules reach the electrode, yet the molecules separate due to electrophoretic mobilities. 

Nucleic acids have negative phosphate backbones. Hence they move towards the anode in DNA electrophoresis. Ampholytes, like proteins, bear both positive and negative charges. Such compounds have negative charge in normal conditions and migrate towards the anode. At the same time, they are positively charged in acidic conditions and move towards the cathode. Hence, protein bears a negative or positive charge depending on solvent pH and isoelectric point.

Factors Affecting the Rate of Ion Mobility

The velocity of ions depends on both inherent factors and the external environment.

Inherent factors

The inherent factors that affect the velocity of ions are:

• Charge density
• Molecular weight
• The net charge of the molecule
• Size and shape of the molecule

External factors

The external factors affecting the rate of movement of ions are:

• Electrical parameters, like current, voltage, and power
• Viscosity and pore size of supporting medium
• Temperature
• The pH of the buffer

Electrophoresis Instrument

Modern electrophoresis equipment and systems vary based on its types and forms. However, all the electrophoretic system possesses two essential components:

Power supply drives the movement of ionic species in the medium and allows adjustment and control of either the current or the voltage.

• An electrophoresis unit

An electrophoretic system depends on its type but essentially consists of two electrodes of opposite charge (anode and cathode), connected by a conducting medium called an electrolyte. In addition, a supportive medium is present in electrophoretic systems like gel and paper electrophoresis.

• Buffer (Electrolyte)

Buffers carry applied electric current and provide appropriate pH for the process. Conducting (running) buffers like Tris borate EDTA (TBE) and Tris-acetate acid EDTA (TAE) are commonly used.

• Supportive Medium

The supportive medium is the matrix (gel), in which biomolecules are separated. It can be in the slab or capillary form. The supportive mediums used are sugar polymers like agarose gel, polyacrylamide gel, starch gel, and cellulose acetate gel. The medium runs either vertical or horizontal gel systems in gel electrophoresis. Horizontal: agarose gel electrophoresis, and vertical: SDS-PAGE. The higher the pore size, the higher the speed traveled by charged particles.

General Procedure of Electrophoresis

The electrophoresis process has three main steps; separation, detection, and quantification.

The instrument set up is according to its type. In the gel electrophoresis, gels are prepared and cast. ( http://www.tntechoracle.com/ ) Then placed into the electrophoresis chamber. The supportive medium can be agarose gels or polyacrylamide gels. Then appropriate buffer solution is added to the system.

After the proper setup of the instrument, the sample is placed into the medium. Then the sample is run at a specific current, voltage, or power.

Detection and Quantification

Staining with a dye or autoradiography (for radioactive samples) helps in the detection of the separated components.

Quantification is done using a densitometer or by direct measurement using an optical detection system. For example, protein is fixed by precipitating in gel with acetic acid. Methanol helps prevent the diffusion of proteins from the gel during the staining process.

Forms of Electrophoresis

Based on the forms, it is of two types; zone and moving boundary electrophoresis.

In the moving boundary electrophoresis , charged molecules migrate in a free-moving solution without a supporting medium. E.g., Capillary electrophoresis.

Types of Electrophoresis

Based on the nature of the supporting medium, it is of the following types:

• Agarose gel electrophoresis
• Polyacrylamide gel electrophoresis
• Cellulose Acetate Electrophoresis
• Capillary Electrophoresis

Depending on the mode of technique, it has the following types:

• Paper electrophoresis
• Isoelectric focusing electrophoresis
• Two-dimensional Polyacrylamide gel electrophoresis
• Pulse field gel electrophoresis
• Immunoelectrophoresis
• Capillary electrophoresis
• High voltage electrophoresis
• Isotachophoresis
• Microchip electrophoresis

Uses of Electrophoresis

It is applied for routine laboratory experiments, disease diagnosis, research-oriented separations and identification. Similarly, it is used in various other fields, like forensics, agriculture, pharmaceutical, foods, etc. Some of its applications are described below:

DNA Analysis and DNA Fragmentation

Gel electrophoresis is the core technique for genetic analysis and purification of nucleic acids for further studies or disease diagnosis.

Identifying Specific protein

• The rate of movement of macromolecules in an electric field is a helpful parameter to know any changes in amino acids regarding their charge.
• Quantitative analysis of specific serum protein classes such as gamma globulins and albumins
• It helps in the identification and quantitation of hemoglobin and its subclasses.
• It also helps in the identification of monoclonal protein in either serum or urine.
• Likewise, it helps in the separation and quantitation of significant lipoprotein classes.
• Immunoelectrophoresis helps to analyze several kinds of protein’s existence and how they behave chemically in different environments.
• It is also helpful in purifying proteins for different purposes.
• Similarly, it is useful in determining the molecular weights of protein.

Coenzymes separation

It is useful in separating and quantifying coenzymes such as creatine, kinase, lactate dehydrogenase, and alkaline phosphatase coenzyme to their respective subtypes.

Analysis of chemical compounds

• It helps analyze compounds, such as water, soil, air quality or contamination, food quality, processing hygiene, and medical forensic analysis.
• It also helps in analyzing transition metals.
• Likewise, it helps to analyze organic compounds.
• Similarly, it helps in analyzing components of pesticides.
• Angerish A (2018). Study of Electrophoresis Techniques and its Types. Journal of Emerging Technologies and Innovative Research. 5(9): 299-304.
• Fritsch, R., & Krause, I. (2003). ELECTROPHORESIS. Encyclopedia Of Food Sciences And Nutrition , 2055-2062. https://doi.org/10.1016/b0-12-227055-x/01409-7
• Chin et al. (2013). Electrophoresis: What does a Century-Old Technology Hold for the Future of Separation Science? International Research Journal of Applied and Basic Sciences. 7(4): 213-221.
• Westermeier, R. (2005). Gel Electrophoresis. Els . https://doi.org/10.1038/npg.els.0005335
• Wilson K & Walker J (1994). Principles and Techniques of Practical Biochemistry: Electrophoretic Techniques. 4 th Ed. Cambridge University Press. 4 th Ed: 425-460.

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Gel electrophoresis of DNA is one of the most frequently used techniques in molecular biology. Typically, it is used in the following: the analysis of in vitro reactions and purification of DNA fragments, analysis of PCR reactions, characterization

Gel electrophoresis of DNA is one of the most frequently used techniques in molecular biology. Typically, it is used in the following: the analysis of in vitro reactions and purification of DNA fragments, analysis of PCR reactions, characterization of enzymes involved in DNA reactions, and sequencing. With some ingenuity gel electrophoresis of DNA is also used for the analysis of cellular biochemical reactions. For example, DNA breaks that accumulate in cells are analyzed by the comet assay and pulsed-field gel electrophoresis (PFGE). Furthermore, DNA replication intermediates are analyzed with two-dimensional (2D) gel electrophoresis. Moreover, several new methods for analyzing various chromosomal functions in cells have been developed. In this chapter, a brief introduction to these is given.

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Capillary Electrophoresis

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Capillary electrophoresis is an analytical technique that separates ions based on their electrophoretic mobility with the use of an applied voltage. The electrophoretic mobility is dependent upon the charge of the molecule, the viscosity, and the atom's radius. The rate at which the particle moves is directly proportional to the applied electric field--the greater the field strength, the faster the mobility. Neutral species are not affected, only ions move with the electric field. If two ions are the same size, the one with greater charge will move the fastest. For ions of the same charge, the smaller particle has less friction and overall faster migration rate. Capillary electrophoresis is used most predominately because it gives faster results and provides high resolution separation. It is a useful technique because there is a large range of detection methods available. 1

Introduction

Endeavors in capillary electrophoresis (CE) began as early as the late 1800’s. Experiments began with the use of glass U tubes and trials of both gel and free solutions. 1 In 1930, Arnes Tiselius first showed the capability of electrophoresis in an experiment that showed the separation of proteins in free solutions. 2 His work had gone unnoticed until Hjerten introduced the use of capillaries in the 1960’s. However, their establishments were not widely recognized until Jorgenson and Lukacs published papers showing the ability of capillary electrophoresis to perform separations that seemed unachievable. Employing a capillary in electrophoresis had solved some common problems in traditional electrophoresis. For example, the thin dimensions of the capillaries greatly increased the surface to volume ratio, which eliminated overheating by high voltages. The increased efficiency and the amazing separating capabilities of capillary electrophoresis spurred a growing interest among the scientific society to execute further developments in the technique.

Instrumental Setup

A typical capillary electrophoresis system consists of a high-voltage power supply, a sample introduction system, a capillary tube, a detector and an output device. Some instruments include a temperature control device to ensure reproducible results. This is because the separation of the sample depends on the electrophoretic mobility and the viscosity of the solutions decreases as the column temperature rises. 3 Each side of the high voltage power supply is connected to an electrode . These electrodes help to induce an electric field to initiate the migration of the sample from the anode to the cathode through the capillary tube. The capillary is made of fused silica and is sometimes coated with polyimide. 3 Each side of the capillary tube is dipped in a vial containing the electrode and an electrolytic solution, or aqueous buffer. Before the sample is introduced to the column, the capillary must be flushed with the desired buffer solution. There is usually a small window near the cathodic end of the capillary which allows UV-VIS light to pass through the analyte and measure the absorbance. A photomultiplier tube is also connected at the cathodic end of the capillary, which enables the construction of a mass spectrum, providing information about the mass to charge ratio of the ionic species.

Electrophoretic Mobility

Electrophoresis is the process in which sample ions move under the influence of an applied voltage. The ion undergoes a force that is equal to the product of the net charge and the electric field strength. It is also affected by a drag force that is equal to the product of \(f\), the translational friction coefficient, and the velocity. This leads to the expression for electrophoretic mobility :

\[ \mu_{EP} = \dfrac{q}{f} = \dfrac{q}{6\pi \eta r} \label{1} \]

where f for a spherical particle is given by the Stokes’ law; η is the viscosity of the solvent, and \(r\) is the radius of the atom. The rate at which these ions migrate is dictated by the charge to mass ratio. The actual velocity of the ions is directly proportional to E, the magnitude of the electrical field and can be determined by the following equation 4 :

\[ v = \mu_{EP} E \label{2} \]

This relationship shows that a greater voltage will quicken the migration of the ionic species.

Electroosmotic Flow

The electroosmotic flow (EOF) is caused by applying high-voltage to an electrolyte-filled capillary. 4 This flow occurs when the buffer running through the silica capillary has a pH greater than 3 and the SiOH groups lose a proton to become SiO - ions. The capillary wall then has a negative charge, which develops a double layer of cations attracted to it. The inner cation layer is stationary, while the outer layer is free to move along the capillary. The applied electric field causes the free cations to move toward the cathode creating a powerful bulk flow. The rate of the electroosmotic flow is governed by the following equation:

\[ \mu_{EOF} = \dfrac{\epsilon}{4\pi\eta} E\zeta \label{3} \]

where ε is the dielectric constant of the solution, η is the viscosity of the solution, E is the field strength, and ζ is the zeta potential. Because the electrophoretic mobility is greater than the electroosmotic flow, negatively charged particles, which are naturally attracted to the positively charged anode, will separate out as well. The EOF works best with a large zeta potential between the cation layers, a large diffuse layer of cations to drag more molecules towards the cathode, low resistance from the surrounding solution, and buffer with pH of 9 so that all the SiOH groups are ionized. 1

Capillary Electroseparation Methods

There are six types of capillary electroseparation available: capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic capillary chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP). They can be classified into continuous and discontinuous systems as shown in Figure 3. A continuous system has a background electrolyte acting throughout the capillary as a buffer. This can be broken down into kinetic (constant electrolyte composition) and steady-state (varying electrolyte composition) processes. A discontinuous system keeps the sample in distinct zones separated by two different electrolytes. 6

Capillary Zone Electrophoresis (CZE)

Capillary Zone Electrophoresis (CZE), also known as free solution capillary electrophoresis, it is the most commonly used technique of the six methods.A mixture in a solution can be separated into its individual components quickly and easily.The separation is based on the differences in electrophoretic mobility, which is directed proportional to the charge on the molecule, and inversely proportional to the viscosity of the solvent and radius of the atom.The velocity at which the ion moves is directly proportional to the electrophoretic mobility and the magnitude of the electric field. 1

T he fused silica capillaries have silanol groups that become ionized in the buffer. The negatively charged SiO - ions attract positively charged cations, which form two layers—a stationary and diffuse cation layer. In the presence of an applied electric field, the diffuse layer migrates towards the negatively charged cathode creating an electrophoretic flow (\(\mu_{ep}\)) that drags bulk solvent along with it. Anions in solution are attracted to the positively charged anode, but get swept to the cathode as well. Cations with the largest charge-to-mass ratios separate out first, followed by cations with reduced ratios, neutral species, anions with smaller charge-to-mass ratios, and finally anions with greater ratios. The electroosmotic velocity can be adjusted by altering pH, the viscosity of the solvent, ionic strength, voltage, and the dielectric constant of the buffer. 1

Capillary Gel Electrophoresis (CGE)

CGE uses separation based on the difference in solute size as the particles migrate through the gel. Gels are useful because they minimize solute diffusion that causes zone broadening, prevent the capillary walls from absorbing the solute, and limit the heat transfer by slowing down the molecules. A commonly used gel apparatus for the separation of proteins is capillary SDS-PAGE . It is a highly sensitive system and only requires a small amount of sample . 1

Micellar Electrokinetic Capillary Chromatography (MEKC)

MEKC is a separation technique that is based on solutes partitioning between micelles and the solvent. Micelles are aggregates of surfactant molecules that form when a surfactant is added to a solution above the critical micelle concentration. The aggregates have polar negatively charged surfaces and are naturally attracted to the positively charged anode. Because of the electroosmotic flow toward the cathode, the micelles are pulled to the cathode as well, but at a slower rate. Hydrophobic molecules will spend the majority of their time in the micelle, while hydrophilic molecules will migrate quicker through the solvent. When micelles are not present, neutral molecules will migrate with the electroosmotic flow and no separation will occur. The presence of micelles results in a retention time to where the solute has little micelle interaction and retention time tmc where the solute strongly interacts. Neutral molecules will be separated at a time between to and tmc. Factors that affect the electroosmotic flow in MEKC are: pH, surfactant concentration, additives, and polymer coatings of the capillary wall. 1

Capillary Electrochromatography (CEC)

The separation mechanism is a packed column similar to chromatography. The mobile liquid passes over the silica wall and the particles. An electroosmosis flow occurs because of the charges on the stationary surface. CEC is similar to CZE in that they both have a plug-type flow compared to the pumped parabolic flow that increases band broadening. 1

Capillary Isoelectric Focusing (CIEF)

CIEF is a technique commonly used to separate peptides and proteins. These molecules are called zwitterionic compounds because they contain both positive and negative charges. The charge depends on the functional groups attached to the main chain and the surrounding pH of the environment. In addition, each molecule has a specific isoelectric point (pI). When the surrounding pH is equal to this pI, the molecule carries no net charge. To be clear, it is not the pH value where a protein has all bases deprotonated and all acids protonated, but rather the value where positive and negative charges cancel out to zero. At a pH below the pI, the molecule is positive, and then negative when the pH is above the pI. Because the charge changes with pH, a pH gradient can be used to separate molecules in a mixture. During a CIEF separation, the capillary is filled with the sample in solution and typically no EOF is used (EOF is removed by using a coated capillary). When the voltage is applied, the ions will migrate to a region where they become neutral (pH=pI). The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). Compounds of equal isoelectric points are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection. 6

Calculating pI

An amino acid with n ionizable groups with their respective pKa values pK 1 , pK 2 , ... pk n will have the pI equal to the average of the group pkas: pI = (pK 1 +pK 2 +...+pk n )/n. Most proteins have many ionizable sidechains in addition to their amino- and carboxy- terminal groups. The pI is different for each protein and it can be theoretically calculated according to the Henderson-Hasselbalch approximation, if we know amino acids composition of protein. In order to experimentally determine a protein's pI 2-Dimensional Electrophoresis (2-DE) can be used. The proteins of a cell lysate are applied to a pH immobilized gradient strip, upon electrophoresis the proteins migrate to their pI within the strip. The second dimension of 2-DE is the separation of proteins by MW using a SDS-gel.

Capillary Isotachorphoresis (CITP)

CITP is the only method to be used in a discontinuous system. The analyte migrates in consecutive zones and each zone length can be measured to find the quantity of sample present. 1

Capillary Electrophoresis versus High Performance Liquid Chromatography (HPLC)

• CE has a flat flow, compared to the pumped parabolic flow of the HPLC. The flat flow results in narrower peaks and better resolution (Figure \(\PageIndex{4}\)).
• CE has a greater peak capacity when compared to HPLC—CE uses millions of theoretical plates.
• HPLC is more thoroughly developed and has many mobile and stationary phases that can be implemented.
• HPLC has more complex instrumentation, while CE is simpler for the operator.
• HPLC has such a wide variety of column lengths and packing, whereas CE is limited to thin capillaries.
• Both techniques use similar modes of detection.
• Can be used complementary to one another.

• Calculate µEP if q= +1, η is 3.7 (lb s/ft 2 ) x 10 -5 and the radius of the atom is 2 nm.
• How does buffer pH affect the capillary?
• How does hydrophilicity affect MEKC?
• What advantages does capillary electrophoresis provide over liquid chromatography?
• Give reasons why “Analyte A” migrated first, while “Analyte D” migrated last.

• Li, Sam. Capillary Electrophoresis: Principles, Practice, and Applications. Journal of Chromatography Library; Elsevier Science Publishers: The Netherlands, 1992; Vol 52.
• Petersen, John R., and Amin A. Mohammad, eds. Clinical and Forensic Applications of Capillary Electrophoresis. New York: Humana P, 2001.
• Camilleri, Patrick. Capillary Electrophoresis. New York: C R C P LLC, 1997.
• Altria, Kevin D., Capillary Electrophoresis Guidebook : Principles, Operation and Applications. New York: Humana P, 1995.
• Landers, James P., Handbook of Capillary Electrophoresis. New York: C R C P LLC, 1996.
• Weston, A.; Brown, P. HPLC and CE: Principles and Practice; Academic Press: San Diego, 1997.

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1.4: PCR and Gel Electrophoresis

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• Marjorie Hanneman, Walter Suza, & Donald Lee
• Iowa State University via Iowa State University Digital Press

Learning Objectives

At the completion of this PCR lesson, learners will be able to:

• List the 5 chemical components of a PCR reaction and describe their roles.
• List the functions of the 3 temperature cycles which are repeated during a PCR reaction.
• Describe the process of observing results and interpreting results of a PCR experiment.
• List possible uses of PCR in genetic testing and in research.

The polymerase chain reaction laboratory technique is used in a variety of applications to make copies of a specific DNA sequence. This lesson describes how a PCR reaction works, what it accomplishes, and its basic requirements for success. Examples of interpreting results are given. PCR’s strengths, weaknesses, and applications to plant biotechnology are explained.

The Discovery of PCR

In 1983, Kary Mullis was driving along a Californian mountain road late one night. As a molecular biologist, Dr. Mullis was imagining a better way to study DNA . This late-night thinking led to a revolutionary way to make laboratory copies of DNA molecules (Saiki et al. 1985, Mullis 1990). In the decades since, the polymerase chain reaction or PCR , has become the standard method used for detecting specific DNA or RNA sequences. Selling the equipment and reagent kits for PCR is a multi-billion-dollar business because DNA and RNA detection is critical information in many applications.

In Vitro vs. In Vivo Replication

PCR is an In Vitro process; a series of chemical reactions that happen outside of a living cell. This laboratory technique is modeled after an In vivo process, the living cell’s natural ability to replicate DNA during normal cell cycles (see Lesson on DNA: The Genetic Material). Every living cell makes a duplicate copy of each chromosome before the cell is ready to divide. Figure 1 below illustrates the key parts of In vivo DNA replication that are the basis for PCR success.

There are other enzymes that play an important role in in vivo replication. However, PCR works as an in vitro DNA replication process by using just one of these enzymes. Mullis imagined a chemical reagent and a temperature change step in the method that could perform the work of the other two enzymes. It should be noted that because Dr. Kerry Mullis had learned about the details of in vivo DNA replication, he could create this science changing in vitro method.

Before reading the description of PCR components and processes, watching this video can help you visualize the importance of each step,

Name and Chemical Components of PCR

The name ‘Polymerase Chain Reaction’ represents the nature of the process. ‘Polymerase’ because DNA Polymerase III is required for constructing new DNA strands, just like in a living cell. ‘Chain Reaction’ describes repeating cycles of replication which target a specific segment of a chromosome and use a “copy the copies” progression each cycle that doubles the amount of DNA copies of a specific segment of DNA present each cycle. In just 20 cycles of the chain reaction, over one million (2 20 ) copies of that specific segment of DNA can be produced. This is enough DNA to see with your naked eye. The goal of PCR is to make millions of copies of a specific segment of DNA that all originate from a single DNA sample.

The five chemical components that must be added to a test tube for the PCR reaction to work, include a DNA template, DNA polymerase III enzyme , single stranded DNA primers, nucleotides, and reaction buffer.

• The DNA template is a sample of DNA that contains the target sequence of DNA for copying.
• DNA pol III. There are two requirements for a suitable DNA polymerase enzyme for PCR. First, the enzyme must have a good activity rate around 75°C. Second, the enzyme should be able to withstand temperatures of 95-100°C without denaturing and losing activity.
• Two primers. Primers are short oligonucleotides of DNA, usually around 20 base pairs in length. Because the purpose of PCR is to amplify a specific section of DNA in the genome, such as a known gene, then primers of specific sequences must be used. The geneticist planning the PCR reaction will design a forward primer to bind to one strand and a reverse primer that complements and binds to the other strand. The primer design process to select forward and reverse primers is requiring appropriate genetics thinking and is describe later in this reading.
• The four different deoxyribonucleotide triphosphates (dNTPs). Adenine (A), guanine (G), cytosine (C), and thymine (T) are needed to provide the building blocks for DNA replication. DNA polymerase will add each complementary base to the new growing DNA strand according to the original strand’s sequence following normal A-T and C-G pairings.
• Finally, a reaction buffer. This creates a stable pH and provides the Mg 2 + cofactor needed for DNA pol III activity.

Three Temperature Cycles

A key insight to the success of PCR as an in vitro DNA replication process which generates millions of specific sequence copies was a three-temperature cycle which accomplish three parts of DNA replication: denaturation of the double stranded template, annealing of the primers to the single strands and extension of new strand synthesis by DNA pol III.

In general, a single PCR run will undergo 25-35 cycles. The first step for a single cycle is the denaturation step, in which the double-stranded DNA template molecule (Figure 2) is made single-stranded (Figure 3) . The temperature for this step is typically in the range of 95-100°C, near boiling. The high heat breaks the hydrogen bonds between the strands but does not break the sugar-phosphate bonds that hold the nucleotides of a single strand together ( Figure 3 ).

Thousands of copies of the single stranded primers and the individual nucleotides were added to the test tube prior to beginning the cycles. Both the primers and nucleotides will become part of the new DNA strands. The second step in the PCR reaction is to cool the temperature in the test tube to 45-55°C. This is the primer annealing step in which the primers bind to complementary sequences in the single-stranded DNA template. The two primers are called the forward and the reverse primer and are designed because their sequences will target the desired segment of the DNA template for replication (Figure 4).

The geneticist planning the PCR analysis must “design” the forward and reverse primers and then buy them from a vendor who can synthesize single stranded DNA that has a specific sequence and length. The two most important criteria for primer design are the following.

• One primer must have a sequence that complements one of the template strands and the other primer must be complementary to the other strand. BOTH strands need to be primed for the replication process.
• The primers must bind so that their 3′ ends are ‘pointing’ in the direction of the other primer. This ensures that the sequence between the primers is replicated in the PCR cycles.

Extension: The final PCR step is when the DNA polymerase enzyme reads the template and connects new nucleotides to the primer’s 3’ end, extending a new complementary strand of DNA (Figure 5). Completion of the final step and the first cycle of PCR, will make two double stranded DNA copies from the original template DNA, doubling of the amount of DNA present. The test tube is heated to around 75°C , optimizing DNA pol. III activity and the newly synthesizing DNA strand is extended as the template strand is read by DNA pol. III . The Extension step will run for a few minutes and this step complet es one PCR cycle.

For cycle 2, the denaturation, annealing, and extension steps are repeated (Figure 6 a, b, c). This time, though there will be twice as many DNA template molecules compared to what there was at the beginning of cycle 1. Copies are being made by reading the original template and copies are made by reading the copies made in the previous cycle. Therefore, if everything is working correctly, the DNA replication in the test tube is a chain reaction where at the end of a cycle, there is double the amount of that DNA sequence as what was found at the beginning of the cycle.

Because thousands of copies of the forward and reverse primer are added at the start of PCR, all the single strand templates, both the original, the copies in cycle 3 and beyond, and the copies of the copies made from previous cycles will be primed for the extension step of the cycles.

Thermal cycler

When PCR was first invented, scientists used water baths set at different temperatures and the hand transfer of test tubes at timed intervals to run the PCR reaction. Once the technique became a proven technology for DNA analysis, engineers went to work to create PCR machines. The instrument which heats and cools the DNA samples is called a thermal cycler (Figure 7). Each small tube or sample well in a plate contains all the chemical components needed for a PCR reaction. Adding a specific sample to the reaction mix provides the template DNA. A thermal cycler can be programmed for specific temperatures and the amount of time spent at each temperature. The engineered design of thermal cyclers to maximize the accurate replication of the targeted DNA in a small sample volume with the minimum amount of time can be critical in many applications of PCR.

Taq DNA polymerase

When Dr. Kerry Mullis ran the first PCR experiments, he needed to add a new sample of DNA pol III after each denaturation step. This was because the high temperature needed to denature the double stranded DNA template also denatured the DNA pol III protein structure. The DNA pol III enzyme commonly available to molecular geneticists was from E. coli bacteria and this enzyme had no stability at near boiling temperatures. Fortunately, biologists had been investigating Thermus aquaticus, (Taq) a thermophilic eubacterium found in hot springs (Chien et al. 1976). The Taq version of DNA pol III does not easily denature in the hot temperatures required in PCR; plus, it has a good efficiency, able to add 60 base pairs/sec at 70°C. Like all other DNA polymerases, Taq DNA pol III cannot begin DNA replication without the addition of a starting primer. Thus, the discovery of Taq DNA pol III and the commercial availability of this enzyme made PCR a more reliable and doable technology which hastened its application to science investigation and diagnostic testing.

Visualizing the Results with Electrophoresis

Once a PCR reaction has been completed, we need to be able to see the results. To do this, a sample of the PCR mixture is loaded into an agarose gel for electrophoresis. The agarose gel contains a matrix of pores which enables it to separate DNA fragments based on their sizes. For details about setting up and running an electrophoresis gel, see Electrophoresis: How Scientist observe fragments of DNA

Figure 8 shows a picture of a gel electrophoresis gel that is running. The box on the right contains DNA loaded in the agarose gel. The gel placed in an aqueous solution of electrolytes. Depending on the type of dye used, color bands are a dye that was added to the PCR sample before it was loaded into the sample well. This allows for the tracking of the DNA’s progression through the gel. Hooked up to this gel unit is an electrical power source which provides the force to move the DNA through the gel. Since DNA molecules are negatively charged, they will migrate towards the red, positive electrode. Shorter DNA fragments move faster than longer fragments through the pores in the gel.

After the gel is run, the DNA is stained with a chemical that binds specifically to DNA molecules and then will either reflect a specific color of visible light or fluoresce a specific color when viewed with ultraviolet light. A single ‘band’ contains 1000s of individual DNA fragments, all of the same length. Figure 10 illustrates the visual information that can be obtained from an electrophoresis gel after it has run. The electric current uniformly moves all the DNA fragments through a gel in the same direction. The sample wells at the top of the gel image thus establish lanes for the DNA samples to move.

Below is a description of what information is revealed from each lane.

• Lane L: This was loaded with the DNA size ladder that contains copies of seven different lengths of DNA fragments. Commercial vendors of the DNA size ladder provide information on the lengths of each fragment in base pairs (bp). Running this lane provides an estimate of the DNA fragment lengths in the sample lanes (1-5).
• Lane 1: The PCR sample loaded in this lane has copies of a single length of DNA. The length is slightly more than 400 base pairs.
• Lane 2: The PCR sample loaded in this lane has copies of two lengths of DNA. One fragment is the same length as the fragments in lane 1 and the second fragment is slightly less than 400 bp.
• Lane 3: The PCR sample loaded in this lane has copies of one fragment that is the same length as the shorter fragments in lane 2.
• Lane 4: The positive control worked as predicted. The sample was set up with all the same reagents as the other PCR samples plus a DNA template that was known to contain the sequence targeted by the PCR primers that would generate a 410 base pair fragment.
• Lane 5: The negative control worked as predicted. The sample was set up with all the same reagents as the other PCR samples except no template DNA was added. Therefore, we would not expect the PCR reaction to work and the absence of a band of DNA fragments is as expected.

Because the positive and negative controls worked as expected, the biologist can be confident that the bands of DNA observed in lanes 1,2 and 3 reveal genetic information about the individual providing that DNA sample.

Advantages of PCR

PCR quickly became the method of choice for many types of DNA analysis because of several advantages over other DNA detection methods. The first; it is a simple procedure to set up and run. Fewer steps save time in getting a DNA analysis result. The second is the sensitivity. A very small amount of template DNA in the sample can be detected. Even just a few skin cells from one human hair contain enough DNA, making PCR useful in forensics. The third is that it can be designed to accurately differentiate genetic samples that are different by as little as single nucleotide in the targeted sequence. Finally, in application where large numbers of samples need to be subjected to the same PCR analysis, automation and robotic assistance allow the processing of many samples in a very short time. For example, an automated PCR would be critical for testing large numbers of people in hours during a pandemic.

Limitations of PCR

There are some drawbacks of using PCR that one should be aware of as well. First, the sequence of the gene or chromosome region being targeted is required. This limitation is rapidly diminishing as gene and genome sequencing technology and sharing of this sequence through Internet data bases has emerged as the norm in genetic analysis. Because of the size of the genomes of living things and a high conservation of gene sequences in many organisms, primers designed for a PCR test must be empirically tested with the proper controls. Biologists can easily generate false positive DNA from PCR that is caused by contamination or lack of specificity in primer design. There may also be a need to optimize concentrations of each chemical component. For example, changing the amount of DNA template, MgCl2 and Taq polymerase can affect both the quantity and quality of bands produced. Some studies have shown that even the brand of Taq polymerase can affect results (Holden et al. 2003). Likewise, the temperature cycles may need to be fine-tuned for a specific PCR test. Finally, as an in vitro DNA replication method, PCR cannot replicate entire chromosomes.

To briefly highlight the topics of this lesson, remember that PCR is a relatively easy lab technique which amplifies the amount of DNA present, much like living cells do in the beginning stages of a cell cycle. There are 5 chemical components of a PCR reaction: a DNA template , a DNA polymerase , primers, nucleotides , and a buffer. The 3 temperature steps for one cycle are the denaturation, primer annealing and extension steps. There are now many variations and uses of PCR ranging from forensics to genomic studies to identifying transgenic crops.

Watch these videos to learn more about PCR:

• Polymerase Chain Reaction (PCR) from DNA Learning Center
• Polymerase Chain Reaction (from UNL)