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Electrophoresis through agarose or polyacrylamide gels is the standard way to separate, identify and purify nucleic acid fragments. The location of the nucleic acid within in the gel can be determined by using the fluorescent intercalating dye ethidium bromide.
Agarose gels have a smaller resolving power than polyacrylamide gels but a greater range of separation - from 200 bp to >50 kb using standard gels and electrophoresis equipment. RNAs up to 10 000 kb can be separated in agarose gels using pulsed field gel electrophoresis.
Polyacrylamide gels have enough resolving power to separate fragments differing by only one base pair in size, but their range is ~ 5 to 1000 bp. They are much more difficult to handle than agarose gels.
Formaldehyde-agarose gel electrophoresis
Agarose is a polysaccharide obtained from seaweed. There are frequently contaminants - other polysaccharides, salts and proteins - and different batches as well as different manufacturers brands vary in the level of contaminants and hence in the performance of the agarose. Agarose can be chemically modified to gel and melt at lower temperatures by the addition of hydroxyethyl groups into the polysaccharide chain.
Agarose gels are cast by completely melting the agarose in the desired buffer and then pouring into a mould to harden. RNA is negatively charged at neutral pH and when an electric field is applied, it migrates towards the anode.
RNA retains much of its secondary structure during electrophoresis unless it is first denatured. The addition of formaldehyde to the agarose gel maintains the RNA in its linear (denatured) form
The rate of migration is determined by :-
Molecular size of RNA
Linear RNA becomes orientated in an electric field in an 'end-on' position and migrates through the matrix of the gel at a rate which is inversely proportional to the log10 of the number of base pairs. Larger molecules migrate more slowly because of greater frictional drag as they try to pass through the gel matrix
Linear RNA of a given size migrates through agarose of different concentrations at different rates given by log m = logmo -KrT, where m is the electrophoretic mobility of the RNA, mo is the free electrophoretic mobility of the RNA, Kr is the retardation coefficient and T is the gel concentration
Conformation of the RNA
RNA molecules which fully or partially retain their secondary structure, migrate at different rates to fully denatured RNAs with the same molecular mass
|% age of agarose (w/v) in gel||Efficient range of separation of linear RNA molecules - bp|
|0.3||5 000 - 60 000|
|0/6||1 000 - 20 000|
|0.7||800 - 10 000|
|0.9||500 - 7 000|
|1.2||400 - 6 000|
|1.5||200 - 3 000|
|2.0||100 - 2 000|
At low voltage, the rate of migration of RNA is proportional to the voltage applied. As the voltage is increased, the mobility of larger molecules increases differentially : the effective range of separation therefore decreases with increasing voltage. For maximum resolution, run agarose gels at no more than 5V / cm (measured between the electrodes, not the gel length).
Base composition of the RNA and temperature of the gel
For agarose, neither of these parameters significantly affect the mobilities of RNA (see polyacrylamide gels)
Presence of intercalating dyes
Ethidium bromide reduces the electrophoretic mobility of linear RNA by about 15%. EtBr has greater affinity for double than single-stranded nucleic acids.
Composition of the electrophoresis buffer
In the absence of ions, RNA migrates very slowly if at all. If a 10 x buffer is used by mistake, electrical conductance is very efficient, the current generates a lot of heat and melt down happens !
Apparatus for agarose gel electrophoresis
Agarose gels are usually run as submerged horizontal slab gels. The resistance of the gel is almost the same as the buffer so a high fraction of the current passes through the gel. If a gel is to be run at high voltage, the volume of buffer should be just enough to submerge the gel and keep it wet. Apparatus used should have the following features :-
- allow easy examination of the gel by UV trans-illumination,
- should be supplied with a variety of combs to form wells of various sizes,
- should have a lid to shield electrical connections,
- allow buffer to be removed completely - important if it contains ethidium bromide and
- should allow recirculation of buffer between anode and cathode.
Polyacrylamide gel electrophoresis
Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N'N'N'N'-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration : usually between 3.5 and 20%. Cross-linking BIS-acrylamide is usually added at a ratio of 2g BIS : 38g acrylamide.
|% age acrylamide (w/v)||Effective range of||Size of RNA co-||Size of RNA co-|
|with BIS at 1:20||separation - bp||migrating with Xylene||migrating with|
|3.5||1 000 - 2 000||460||100|
|5.0||80 - 500||260||65|
|8.0||60 - 400||160||45|
|12.0||40 - 200||70||20|
|15.0||25 - 150||60||15|
|20.0||6 - 100||45||12|
Polyacrylamide gels are poured between two glass plates held apart by spacers of 0.4 - 1.0 mm and sealed with tape. Most of the acrylamide solution is shielded from oxygen so that inhibition of polymerisation is confined to the very top portion of the gel. The length of the gel can vary between 10 cm and 100 cm depending on the separation required. They are always run vertically with 0.5 / 1 x TBE as a buffer.
Three main advantages over agarose gels
Resolving power is such that RNA molecules differing in length by 1 base in 500 ie 0.2% can be effectively separated
They can hold up to 10 mg of RNA per slot (20 mg of RNA) without loss of resolution, a much larger amount than agarose gels
The recovered RNA or RNA is extremely pure.
Two types of polyacrylamide gel in general use
Non-denaturing gels : these are run at low voltages - 8V/cm - and 1 x TBE to prevent denaturation of small fragments of RNA by the heat generated in the gel during electrophoresis. The rate of migration is approximately inversely proportional to log10 of their size. However, the base sequence composition can alter the electrophoretic mobility of RNAs such that two RNAs of the same size may show up to a 10% difference in electrophoretic mobility
Denaturing gels : these gels are polymerised with a denaturant that suppresses base pairing in nucleic acids - this is usually urea but can be formamide. Denatured RNA migrates through the gel at a rate which is almost completely independent of its composition or sequence. These gels are used for the analysis of sequencing reactions, RNase protection assays and purification of radiolabelled RNA and RNA probes.
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