Agarose and Polyacrylamide Gel Electrophoresis Matrices

Introduction

Fig: Polyacrylamide Gel Electrophoresis (PAGE)

During the last years molecular biology techniques, such as polymerase chain reaction (PCR), have become widely used for medical and forensic applications, as well as research, and detection and characterization of infectious organisms. In the virology field, it has been demonstrated that the employment of PCR technique offers the advantages of high sensitivity and reproducibility in viral genomic detection and strains characterization. However, the sensitivity in the detection of DNA fragments is also linked to the sensitivity of the electrophoresis matrix applied for PCR product development.

Electrophoresis through agarose or polyacrylamide gels is a standard method used to separate, identify and purify nucleic acids since both these gels are porous in nature. In this blog post, the evaluation of the sensitivity of agarose and polyacrylamide gel electrophoresis matrices in the detection of PCR products is analyzed. For this purpose, rotavirus PCR amplicons were used as a model. Human rotaviruses have been recognized as the most common cause of dehydrating diarrhea in infants and young children on a worldwide scale. These viruses are characterized by the presence of 11 segments of double-stranded RNA surrounded by three separate shells, the core, inner capsid and outer capsid. Currently, rotaviruses are dual classified into G and P genotypes according to the differences of VP7 and VP4 neutralization antigens which form the outer capsid of the virion. Two rotavirus vaccines have been licensed in the year 2006 in many countries. Although large-scale safety and efficacy studies of both rotavirus vaccines have shown excellent efficacy against severe rotavirus gastroenteritis (Ruiz-Palacios et al., 2006; Matson, 2006), the lack of clear data about the protection against genotypes not included in the vaccine formulations underlines the importance of virological surveillance, rotavirus strain characterization and the evaluation of the impact of these vaccines in diminishing the diarrhea illness in our region (Gentsch et al., 2005; Perez-Schael et al., 1990; Velazquez et al., 1996). In addition, the presence of multiple G and/or P genotypes in individual specimens may offer a unique environment for mixed infection acquisition and thereby for the reassortment of rotavirus genes. This could affect both, rotavirus evolution and efficacy performance of current and future vaccines. In this context, knowledge of both the rotavirus genotypes circulating in a community and the incidence of rotavirus mixed infections is essential for acquiring an in-depth understanding of the ecology and distribution of rotavirus strains and anticipating antigenic changes that could affect vaccine effectiveness. For this purpose, rotavirus G and P genotypes are determined by extraction of the viral RNA from fecal specimens followed by analysis by semi-nested reverse-transcriptase PCR (RT-PCR) with primers specific for regions of the genes encoding the VP7 or VP4. The genotype-specific PCR products are then analyzed on an agarose or polyacrylamide gel followed by ethidium bromide staining or silver staining, respectively. The matrix used for electrophoresis should have adjustable but regular pore sizes and be chemically inert, and the choice of which gel matrix to use depends primarily on the sizes of the fragments being separated (Guilliatt, 2002). As commented before, although the importance of specificity and sensitivity of PCR is well known, the mechanism by which the results are measured is equally important (Wildt et al., 2008).

General characteristics of agarose and polyacrylamide matrices Agarose gel electrophoresis (AGE)

Agarose is a natural linear polymer extracted from seaweed that forms a gel matrix by hydrogen-bonding when heated in a buffer and allowed to cool. For most applications, only a single-component agarose is needed and no polymerization catalysts are required. Therefore, agarose gels are simple and rapid to prepare (Chawla, 2004). They are the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation but a relatively low resolving power, since the bands formed in the gels tend to be fuzzy and spread apart. This is a result of pore size and cannot be largely controlled. These and other advantages and disadvantages of using agarose gels for DNA electrophoresis are summarized here

Advantages

• Nontoxic gel medium
• Gels are quick and easy to cast
• Good for separating large DNA molecules
• Can recover samples by melting the gel, digesting with enzyme agarose or treating with chaotropic salts

Disadvantages

• High cost of agarose
• Fuzzy bands
• Poor separation of low molecular weight samples

Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gels are chemically cross-linked gels formed by the polymerization of acrylamide with a crosslinking agent, usually N,N’-methylenebisacrylamide. The reaction is a free radical polymerization, usually carried out with ammonium persulfate as the initiator and N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst. Although the gels are generally more difficult to prepare and handle, involving a longer time for preparation than agarose gels, they have major advantages over agarose gels. They have a greater resolving power, can accommodate larger quantities of DNA without significant loss in resolution and the DNA recovered from polyacrylamide gels is extremely pure (Guilliatt, 2002). Moreover, the pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers. Anyway, it should be noted that polyacrylamide is a neurotoxin (when unpolymerized), but with proper laboratory care, it is no more dangerous than various commonly used chemicals (Budowle & Allen, 1991). Some advantages and disadvantages of using polyacrylamide gels for DNA electrophoresis.

Advantages

• Stable chemically cross-linked gel
• Sharp bands
• Good for separation of low molecular weight fragments
• Stable chemically cross-linked gel

Disadvantages

• Toxic monomers
• Gels are tedious to prepare and often leak
• Need new gel for each experiment

Gel concentration

Agarose gel concentration

The percentage of agarose used depends on the size of fragments to be resolved. The concentration of agarose is referred to as a percentage of agarose to the volume of buffer (w/v), and agarose gels are normally in the range of 0.2% to 3% (Smith, 1993). The lower the concentration of agarose, the faster the DNA fragments migrate. In general, if the aim is to separate large DNA fragments, a low concentration of agarose should be used, and if the aim is to separate small DNA fragments, a high concentration of agarose is recommended

Concentration of agarose (%) DNA size range (bp)

Concentration of agarose (%)	DNA size range (bp)
0.2 0.4 0.6 0.8 1 1.5 2 3	5000-40000 5000-30000 3000-10000 1000-7000 500-5000 300-3000 200-1500 100-1000

The choice of acrylamide concentration is critical for optimal separation of the molecules (Hames, 1998). Choosing an appropriate concentration of acrylamide and the crosslinking agent, methylenebisacrylamide, the pore sized in the gel can be controlled. With increasing the total percentage concentration (T) of monomer (acrylamide plus cross-linker) in the gel, the pore size decreases in a nearly linear relationship. Higher percentage gels (higher T), with smaller pores, are used to separate smaller molecules. The relationship of the percentage of the total monomer represented by the crosslinker (C) is more complex. Researchers have settled on C values of 5% (19:1 acrylamide/bisacrylamide) for most forms of denaturing DNA and RNA electrophoresis, and 3.3% (29:1) for most proteins, native DNA and RNA gels. For optimization, 5% to 10% polyacrylamide gels with variable cross-linking from 1% to 5% can be used. Low cross-linking (below 3% C) yields “long fiber gels” with an increased pore size (Glavač & Dean, 1996). Moreover, it should be pointed out that at low acrylamide/bisacrylamide concentrations the handling of the gels is difficult because they are slimy and thin. Table 4 gives recommended acrylamide/bisacrylamide ratios and gel percentages for different molecular size ranges 

Polyacrylamide gel concentration

Acrylamide/Bis Ratio	Gel %	Native DNA/RNA (bp)	Denatured DNA/RNA (bp)
19:1	4 6 8 10 12	100-1500 60-600 40-500 30-300 20-150	70-500 40-400 20-200 15-150 10-100
29:1	5 6 8 10 12 20	200-2000 80-800 60-400 50-300 40-200 <40	70-800 50-500 30-300 20-200 15-150 <40

Polyacrylamide gel concentration for resolving DNA/RNA molecules. 

Note: Recommended applications for each formulation are shown in bold.

Electrophoretic buffer systems

Effective separation of nucleic acids by agarose or polyacrylamide gel electrophoresis depends upon the effective maintenance of pH within the matrix. Therefore, buffers are an integral part of any electrophoresis technique. Moreover, the electrophoretic mobility of DNA is affected by the composition and ionic strength (salt content) of the electrophoresis buffer (Somma & Querci, 2006). Without salt, electrical conductance is minimal and DNA barely moves. In a buffer of high ionic strength, electrical conductance is very efficient and a significant amount of heat is generated. Different categories of buffer systems are available for electrophoresis: dissociating and non-dissociating, continuous and discontinuous.

Dissociating and non-dissociating buffer systems

The electrophoretic analysis of single stranded nucleic acids is complicated by the secondary structures assumed by these molecules. Separation on the basis of molecular weight requires the inclusion of denaturing agents, which unfold the DNA or RNA strands and remove the influence of shape on their mobility. Nucleic acids form structures stabilized by hydrogen bonds between bases. Denaturing requires disrupting these hydrogen bonds. The most commonly dissociating buffer systems used include urea and formamide as DNA denaturants. Denatured DNA migrates through these gels at a rate that is almost completely dependent on its base composition and sequence. Denaturing or dissociating buffer systems for proteins include the use of sodium dodecyl sulfate (SDS). In the SDS-PAGE system, developed by Laemmli (1970), proteins are heated with SDS before electrophoresis so that the charge-density of all proteins is made roughly equal. Heating in SDS, an anionic detergent, denatures proteins in the samples and binds tightly to the uncoiled molecule (with net negative charge). Consequently, when these samples are electrophoresed, proteins separate according to mass alone, with very little effect from compositional differences. DNA molecules are negatively charged; therefore the addition of SDS in the gel preparations is only with the aim of enhancing the resolution power of the bands (Day & Humphries, 1994). In the absence of denaturants, double stranded DNA (dsDNA), like a PCR product, retains its double helical structure, which gives it a rodlike form as it migrates through a gel. During the electrophoresis of native molecules in a non-dissociating buffer system, separation takes place at a rate approximately inversely proportional to the log10 of their size.

Continuous and discontinuous buffer systems

In the continuous buffer systems, the identity and concentration of the buffer components are the same in both the gel and the tank. Although continuous buffer systems are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels. These buffer systems are used for most forms of DNA agarose gel electrophoresis, which commonly contain EDTA (pH 8.0) and Trisacetate (TAE) or Tris-borate (TBE) at a concentration of approximately 50mM (pH 7.5-7.8). TAE is less expensive but not as stable as TBE. In addition, TAE gives better resolution of DNA bands in short electrophoretic separations and is often used when subsequent DNA isolation is desired. TBE is used for polyacrylamide gel electrophoresis of smaller molecular weight DNA (MW<2000) and agarose gel electrophoresis of longer DNA where high resolution is not essential. Discontinuous (multiphasic) systems employ different buffers for tank and gel, and often two different buffers within the gel. Discontinuous systems concentrate or “stack” the samples into a very narrow zone prior to separation, which results in improved band sharpness and resolution. The gel is divided into an upper “stacking” gel of low percentage of acrylamide and low pH (6.8) and a separating gel with a pH of 8.8 and much smaller pores (higher percentage of acrylamide). The stacking gel prevents any high-molecular-weight DNA present in the sample from clogging the pores at the top of the running gel before low molecular weight DNA has entered. Both, the stacking and the separating gels, contain the only chloride as the mobile anion, while the tank buffer contains glycine as its anion, at a pH of 8.8. The major advantage of the discontinuous buffer system over continuous buffer system is that this gel system can tolerate larger sample volumes (Rubin, 1975).

Loading buffer

This is the buffer to be added to the DNA fragment that will be electrophoresed. This buffer contains glycerol or sucrose to increase the density of the DNA solutions; otherwise, the samples would dissolve in running buffer tank and not sink into the gel pocket. The gel loading buffer also contains dyes that facilitate observation of the sample during gel loading and electrophoresis, such as bromophenol blue or xylene cyanol. Because these molecules are small, they migrate quickly through the gel during electrophoresis, thus indicating the progress of electrophoresis (Chawla, 2004). The components and concentrations of the 6X loading dye usually used are: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol; or 0.25% bromophenol blue, 50 mM EDTA, 0.4% sucrose.

Voltage/current applied

The higher the voltage/current, the faster the DNA migrates. If the voltage is too high, band streaking, especially for DNA≥12-15kb, may result. Moreover, high voltage causes a tremendous increase in buffer temperature and current in very short time. The high amount of the heat and current built up in the process leads to the melting of the gel, DNA bands smiling, decrease of DNA bands resolution and fuse blowout. Therefore, it is highly recommended not exceed 5-8 V/cm and 75 mA for standard size gels or 100 mA for minigels. On the other side, when the voltage is too low, the mobility of small (≤1kb) DNA is reduced and band broadening will occur due to dispersion and diffusion.

Visualizing the DNA

After the electrophoresis has been completed there are different methods that may be used to make the separated DNA species in the gel visible to the human eye. 7.1 Ethidium bromide staining (EBS) The localization of DNA within the agarose gel can be determined directly by staining with low concentrations of intercalating fluorescent ethidium bromide dye under ultraviolet light. The dye can be included in both, the running buffer tank and the gel, the gel alone, or the gel can be stained after DNA separation. For a permanent record, mostly instant photos are taken from the gels in a dark room. It is important to note that ethidium bromide is a potent mutagen and moderately toxic after an acute exposure. Therefore, it is highly recommended to handle it with considerable caution.

Silver staining (SS)

Silver staining is a highly sensitive method for the visualization of nucleic acid and protein bands after electrophoretic separation on polyacrylamide gels. Nucleic acids and proteins bind silver ions, which can be reduced to insoluble silver metal granules. Sufficient silver deposition is visible as a dark brown band on the gel. All silver staining protocols are made of the same basic steps, which are: i) fixation to get rid of interfering compounds, ii) silver impregnation with either a silver nitrate solution or a silver-ammonia complex solution, iii) rinses and development to build up the silver metal image, and iv) stop and rinse to end development prior to excessive background formation and to remove excess silver ion (Chevallet et al., 2006)