AGAROSE GEL ELECTROPHORESIS OF PLASMID DNA

AIM : To separate and visualize the plasmid DNA by gel electrophoresis.

THEORETICAL BACKGROUND :

Agarose gel electrophoresis is the most suitable physical method of determining size of DNA. During this procedure, DNA is forced to migrate along a cross-linked agarose matrix in response to electric current. DNA contains phosphate groups that confer an overall negative charge to it; and hence, it migrates towards a positive electrode. Many factors determine the migration rate of DNA in a gel, i.e. size of DNA, conformation of DNA, agarose concentration, voltage applied, presence of ethidium bromide, type of agarose used and ionic strength of the running buffer. Electrophoresis is usually a sieving process and the higher the size of DNA it entangles in gel quite easily and migrates slowly. On the other hand, smaller fragments move rather quickly than the larger fragments proportional to their size. The gel matrix can be adjusted by increasing or decreasing the concentration of the gel and a standard 1 % agarose gel can resolve DNA from 0.2 to 30 kb in size.

 

Agarose is mostly isolated from the sea weed of genera Gelidium and Gracilaria and consists of repeated agarobiose, i.e. D-galactose and 3,6-anhydro-L-galactopyranose subunits. In the course of gelation, agarose polymers associate non-covalently and form a network of bundles whose pore size determines the gel's molecular sieving properties. Agarose gels are easy to cast and handle in comparison to the other matrices as the gel setting is a physical change rather than a chemical change as well as samples can be easily recovered from the gel, resulting gels can be stored in plastic bags in a refrigerator.

When heated, agarose solution becomes gel with pore size ranging from 50 to 200 nm. With the addition of fluorescent dyes like ethidium bromide (latex permeable, hence use nitrile gloves), gold view, SYBR Safe, Methylene blue, Crystal violet, Gel red, EZ Vision, Nile Red A, etc. DNA can be visualised under UV detector.

Most of the agarose gels are prepared between 0.7 and 2 % of agarose. A 0.7 % good separation for large DNA fragments, i.e. 5-10 kb, and a 2 % gel shows good resolution for smaller fragments with size ranging from 0.2 to 1 kb.

Low percentage gels are very weak but high percentage gels are usually brittle and do not set evenly. In a gel, the distance between DNA bands of a given length is determined by the percentage of agarose. Percentage of gel is the best way to control the resolution of agarose gel electrophoresis.

When charged molecules are placed in an electric field, they tend to migrate towards either the positive or negative pole as per their charge. In contrast to proteins, which have either a net positive or negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone and hence, migrate towards the anode. The nucleotides in a DNA molecule are linked together by negatively charged phosphodiester groups. For every base pair (average molecular weight of approximately 660), there are two charged phosphate groups. Hence, every charge in DNA molecule is accompanied by approximately the same mass.

When a voltage is applied across the electrodes, a potential gradient is generated. When, this potential gradient is applied, a force is generated on the charged molecule. This force drags the charged molecule towards the electrode. However, there is a frictional force exists that slows down the moment of charged molecules which is the function of the hydrodynamic size of the molecule, the shape of the molecule, the pore size of the medium where electrophoresis is taking place and the viscosity of the buffer.

Reagents and Their Role :

Agarose :

It is used for the electrophoretic separation of nucleic acids. The purest form of agarose is free of DNase and RNase activities. Molecular biology grade agarose is the standard one for the resolution of DNA fragment in the range of 50 bp-50 kb, with the possibility of subsequent DNA extraction from the gel for further analysis. The agarose polymer contains charged groups, in particular pyruvate and sulphate. These negative charged groups can retard the movement of DNA in a process called electroendosmosis (EEO), and low EEO agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids.

Tris-acetate Buffer :

The electrophoretic mobility of DNA is dependent on the composition and ionic strength of the electrophoresis buffer. During the absence of ions, there will be a minimal electrical conductance and DNA migrates slowly. The buffer of high ionic strength and high electrical conductance is efficient and additionally a significant amount of heat is generated. Thus, worsening the situation the gel melts and DNA is denatured. Several different buffers have been recommended for use in electrophoresis of native double stranded DNA. These buffers contain EDTA (pH 8.0) and Trisacetate (TAE), Tris-borate (TBE) or Tris-phosphate (TPE) at an approximate concentration of 50 mM (pH 7.5-7.8). These buffers are generally prepared as concentrated solutions and stored at room temperature, when used the working solution is prepared as 1X.

TAE and TBE are the most commonly used buffers and two of them have their own advantages and disadvantages. Borate has disadvantages as it polymerizes and interacts with cis diols found in RNA. On the other hand, TAE has lowest buffering capacity but it provides the best resolution for larger DNA which implies the need for the lower voltage and more time with a better product. Lithium borate is a relatively new buffer and is ineffective in resolving fragments larger than 5 kb.

10X Stock of Tris Acetate buffer

Prepare a 10X stock solution in 1000 milli-Q water: 48.4 g Tris base, 11.4 ml glacial acetic acid, 20 ml of 0.5 M EDTA or 3.7 g EDTA disodium salt. Dissolve all in 800 ml deionized water and make up the volume to 1000 ml. Store in room temperature and dilute it to 1X prior to use.

Ethidium Bromide :

Ethidium bromide is a fluorescent dye that intercalates between nucleic acid bases and eases the detection of nucleic acid fragments in gel.

When exposed to ultra violet light, the dye flouresces with an orange colour, intensifying 20-fold after binding to DNA. The absorption maxima of EtBr in aqueous solution is between 210 and 285 nm that corresponds to the UV light. Hence, as a result of this excitation EtBr emits orange light with a wave length of 605 nm. EtBr binds with DNA and slips in between its hydrophobic base pairs and stretches the DNA fragment, thus removing the water molecules from ethidium cation. This dehydrogenation results in the increase in fluorescence of the ethidium. However, EtBr is a potential mutagen, suspected carcinogen and it can irritate eyes, skin, mucous membranes and upper respiratory tract at higher concentrations, it is due to the fact that, EtBr intercalates into double stranded DNA, deform the molecules thus blocking the biological processes involving nucleic acids like DNA replication and transcription. Hence, there are many alternatives regarded as less dangerous and with better performance like SYBR dyes.

Gel Loading Buffer :

Loading buffer is mixed with the DNA samples to be used in agarose gel electrophoresis. The dye present in the buffer is used primarily to assess how fast the samples are running during electrophoresis and to render a higher density to the samples than that of the running buffer. The increased density can be achieved by the addition of materials like Ficoll (polysucrose), sucrose or glycerol. There are many colour combinations available to trace the migration rate of the DNA samples. The dyes include Xylene cyanol, Cresol red, Bromophenol blue, Orange G, Tartrazine etc. To prepare a 6X gel loading buffer containing glycerol and bromophenol blue, add 3 ml glycerol (30 %), 25 mg bromophenol blue (0.25 %) and 7 ml of milli-Q water , store at 4°C. The working concentration of the loading buffer should be 1X.

DNA Marker :

A molecular weight size marker is also called a DNA ladder is a set of standards that is used to determine the approximate size of a test molecule during agarose gel electrophoresis. DNA ladders are prepared by two ways : partial ligation, where a 100 bp of DNA piece is partially ligated that gives rise to dimers of 200 bp, trimers of 300 bp, tetramers of 400 bp, pentamers 500 bp and so on. Secondly, restriction digestion of a known DNA sequence by a particular restriction enzyme that gives rise to DNA pieces of varied molecular masses.

DNA Sample :

5 ul of amplified product is sufficient to be visualised after agarose gel electrophoresis. It can be mixed with 1ul of 6X gel loading dye to be loaded in the wells prepared in agarose gel.

Ultraviolet (UV) treatment can cause dose-dependent incision in the sugar phosphate backbone of DNA, and the plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest

- "Nicked Open-Circular" DNA has one strand cut.

- "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo.

- "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed).

- "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.

- "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.