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Relative rates of migration of circular, linear and supercoiled DNA in agarous gel electrophoresis

Relative rates of migration of circular, linear and supercoiled DNA in agarous gel electrophoresis


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I have read the supercoiled plasmid DNA migrates fastest when subjected to agarose gel electrophoresis and this makes sense to me because it is small and experiences the least friction because of its supercoiled structure.

Linear DNA migrates faster than the circular form. I didn't understand why exactly. Shouldn't the circular one be considered to be smaller than the linear, and thus quicker?


First let me supply an illustration of the situation described in the question, together with a reference.

Although you can see this sort of thing, just by searching for “plasmid migration on agarose gel”, this is one of my own from the last millennium (plasmid pBR322), appearing in a text from the last millennium, Adams et al. The Biochemistry of the Nucleic Acids (11th ed).

The poster's attempt to rationalize the results, though expressing the quandary, is marred by the loose use of the word small, and the absence of any statement as to why 'small' pieces of DNA should migrate more rapidly.

The different forms of the plasmid have exactly the same molecular mass, so what is meant by small? My own initial way of looking at this is to consider the agarose gel as a mesh or sieve through which the molecules must pass, so that the larger the volume they occupy in space, the less likely they are to pass through the 'holes' in the mesh. The volume that they occupy in space might be regarded as that generated by rotating them from their midpoint in three dimensions. On this basis the supercoils, in which the DNA is collapsed into a short fat pseudo-linear shape should occupy the least volume in space, and the long thin linear the greatest. The diameter of the circle should place it somewhere between the other two.

So, we reach the same conclusion as the poster: the faster migration of the 'linears' than the nicked circles does not fit this simple model. In the text accompanying the figure I wrote “… a result that is a little unexpected” without attempting to explain it.

Am I able to explain this somewhat unexpected result now? An internet search produces either hand-waving or evasion (I stand to be corrected on this), so that I can only imagine that the result has something to do with the linear molecules having a greater chance of their ends being directed to the holes in the 'mesh' than the nicked circles, because of the open nature of the latter. One can perhaps imagine an hour-glass shaped portion of the sphere for the rotation of the linears that allows the ends to be directed at the holes.

More waving of hands? Perhaps. But the main point is that the physical explanation for the migration of molecules of different shapes during electrophoresis is more complex than a mere biologist might have hoped.


Effects of Supercoiling in Electrophoretic Trapping of Circular DNA in Polyacrylamide Gels

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5–22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 Å, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E −2 ± 0.1 with the field, as opposed to E −1.1 ± 0.1 for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density σ = −0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.


How do I open and configure the Simulate Agarose Gel dialog?

Open the "Simulate Agarose Gel" Dialog

To open the Simulate Agarose Gel dialog, click Tools → Simulate Agarose Gel. .

Select Lane 1, and then choose a MW marker from the drop-down menu.

To choose a lane, click a number above the gel.

Alternatively, click the lane number in the list below the gel.

Specify the Sequence for Digestion or PCR

To specify the sequence for digestion or PCR, expand the Lane [N] menu, then choose the desired file.

New for SnapGene 5.2 and later: SnapGene accurately simulates relative migration rates of supercoiled (covalently closed circular) DNA.

Prior to specifying digestion with restriction enzymes, SnapGene will simulate the migration of a circular sequence as supercoiled DNA. Uncut supercoiled sequences will be marked as such in the list.

The newly added sequence will be displayed in the gel.

Optionally Specify the Gel Buffer

The migration of supercoiled DNA relative to linear DNA differs significantly depending on the electrophoresis buffer.

If you wish to change the electrophoresis buffer used for simulating the migration of supercoiled DNA, click the blue buffer indicator to open SnapGene Preferences.


Figure 2:

Now let see some real images.

The gel image above is the result of restriction digestion. Lane 3, 5, 7, and 8 are a homozygous normal allele with a 184bp band here one band of 68bp is also present, but it is not visible.

Lane 2 is a mutant uncut allele of 252bp.

Lane 1 and 6 are heterozygous contain three alleles: 252bp, 184bp and 68bp. However, it is difficult to distinguish the 64bp band, because, the concentration of gel lower than 3%.

If the concentration of gel was 3%, more sharpen bands will be seen and maybe the 64bp band will appear.

Lane 4 is a molecular marker.


How to run the agarose gel?

The gel matrix is cast as a horizontal slab. Indentations are created using plastic combs in which the DNA is loaded. However, before the DNA is loaded, a loading dye is used to mix the DNA to weigh down the sample in the solution. This method leaves a visible marker, which will help in tracking down the progression of the DNA movement.

For unknown samples, they are run along the DNA ladder with a known DNA length for comparison. The agarose gel is placed in a container (gel tank/box) containing a conductive pH-controlled buffer solution. An electrical field is applied along the length of the gel.

The voltage gradient affects the movement speed through the gel. A DC power supply is used to power the electrical field. In the process of agarose gel electrophoresis, the power supply is set to a constant voltage with consideration to the tank size. (6, 7, and 8)

Image 4: A genomic DNA, which derived from a blood sample, undergone the process of agarose gel electrophoresis.
Picture Source:
researchgate.net

Visualization of DNA

Once the DNA has completely migrated through the gel, the next important step is visualization. The goal is to check for the length and number of molecules in a given sample. The DNA is stained for easy visualization given the fact that DNA is not visible to the naked eye. When imaging gels, the fluorescent stain is used as it provides better detection levels.

The agarose gel image is captured using high sensitivity cameras equipped with blue light transilluminators. It is used to excite the fluorescent stains to emit light and will then be captured by the camera.

The gel docs have some sort of illumination source a filter that gets rid of background light, and a camera to check the signal. The captured image is used to check it according to the DNA band patterns, which is useful in checking the DNA’s length and quantity. (5, 7, 8, and 9)

Image 5: An agarose gel electrophoresis is one of the commonly used procedures for separating biological molecules.
Picture Source:
wikimedia.org

Why agarose is used in gel electrophoresis instead of agar?

Agar is one of the commonly used substances when doing electrophoresis. However, the use of agarose gel is becoming increasingly popular. In fact, it is preferred over agar because it is easy to cast and has fewer charged groups.

It is ideal for separating DNA of various sizes, especially the ones commonly encountered in the laboratory. The separated DNA can be easily viewed with staining and with the help of proper lighting such as the ultraviolet light.

The extraction process is relatively easy too. The typical agarose gel used ranges between 0.7% and 2% and dissolved in an electrophoresis buffer. (2, 4, and 7)


Relative rates of migration of circular, linear and supercoiled DNA in agarous gel electrophoresis - Biology

Recombinant DNA


What is gel electrophoresis?


Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties.

A gel is a colloid in a solid form. The term electrophoresis describes the migration of charged particle under the influence of an electric field. Electro refers to the energy of electricity. Phoresis, from the Greek verb phoros, means "to carry across." Thus, gel electrophoresis refers to the technique in which molecules are forced across a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the driving force. A molecule's properties determine how rapidly an electric field can move the molecule through a gelatinous medium.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

There are two basic types of materials used to make gels: agarose and polyacrylamide. Agarose is a natural colloid extracted from sea weed. Agarose is a chain of sugar molecules, and is extracted from seaweed. Manufacturers prepare special grades of agarose for scientific experimentation. Because the agarose undergoes much commercial processing it is very expensive.

It is very fragile and easily destroyed by handling. Agarose gels have very large "pore" size and are used primarily to separate very large molecules wiht a molecular mass greater than 200 kdal. Agarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior. That is, the bands formed in the agarose gels are fuzzy and spread far apart. This is a result of pore size and it cannot be controlled.

Agarose is a linear polysaccharide (average molecular mas about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%.

Agarose gels are formed by suspending dry agarose in aqueous buffer, then boiling the mixture until a clear solution forms. This is poured and allowed to cool to room temperature to form a rigid gel.


Polyacrylamide

There are two basic types of materials used to make gels: agarose and polyacrylamide. The polyacrylamide gel electrophoresis (PAGE) technique was introduced by Raymond and Weintraub (1959). Polyacrylamide is the same material that is used for skin electrodes and in soft contact lenses. Polyacrylamide gel may be prepared so as to provide a wide variety of electrophoretic conditions. The pore size fo the gel may be varied to produce different molecular seiving effects for separating proteins of different sizes. In this way, the percentage of polyacrylmide can be controlled in a given gel. By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2,000 kdal. This is the ideal range for gene sequencing, protein, polypeptide, and enzyme analysis. Polyacrylamide gels can be cast in a single percentage or with varying gradients. Gradient gels provide continuous decrease in pore size from the top to the bottom of the gel, resulting in thin bands. Because of this banding effect, detailed genetic and molecular analysis can be performed on gradient polyacrylamide gels. Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels.

Migration of DNA Fragments in Agarose

Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.

Additionally, several factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to advantage in optimizing separation of DNA fragments. Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.

Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Similarly, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

Agarose gels, as discussed above provide the most commonly-used means of isolating and purifying fragments of DNA, which is a prerequisite for building any type of recombinant DNA molecule.

By varying buffer composition and running conditions, the utility of agarose gels can be extended. Examples include:

* Pulsed field electrophoresis is a technique in which the direction of current flow in the electrophoresis chamber is periodically altered. This allows fractionation of pieces of DNA ranging from 50,000 to 5 millon bp, which is much larger than can be resolved on standard gels.

* Alkaline agarose gels are prepared with and electrophoresed in buffers containing sodium hydroxide. Such alkaline conditions are useful for analyzing single-stranded DNA.


Agarose Gel Electrophoresis

It is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.


Several additional factors have important effects on the mobility of DNA fragments in agarose gels, and can be used to your advantage in optimizing separation of DNA fragments. Chief among these factors are:

Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.

The image to the right shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. Notice how the larger fragments are much better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.


Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).

Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.

Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.


Question : Electrophoresis can resolve molecules of different sizes. Large molecules migrate more slowly than small molecules in agarose and polyacrylamide gels. However, the fact that nucleic acids of the same length may exist in a variety of conformations can often complicate the interpretation of electrophoretic separations. For instance, individual bacterial

Electrophoresis can resolve molecules of different sizes. Large molecules migrate more slowly than small molecules in agarose and polyacrylamide gels.

However, the fact that nucleic acids of the same length may exist in a variety of conformations can often complicate the interpretation of electrophoretic separations. For instance, individual bacterial plasmids may exist in three forms:

  • Superhelical/supercoiled (Form I):Form I is compact and very tightly coiled, with both DNA strands continuous.
  • Nicked/open circle (Form II): Form II exists as a loose circle because one of the two DNA strands has been broken, releasing the supercoil.
  • Linear (Form III)

All three forms have the same mass, but each will migrate at a different rate through a gel.


What causes faint bands in gel electrophoresis?

The gel matrix acts as a sieve: smaller DNA molecules migrate faster than larger ones, so DNA molecules of different sizes separate into distinct bands during electrophoresis. More DNA in a band gives more intense staining of that band.

Subsequently, question is, what errors could lead to not having a band on the gel after electrophoresis? You may have ran your DNA out of the gel by running for too long. You may have melted your gel by running for too long or using a stale electrophoresis buffer with a low buffer capacity. You may have used a terribly wrong percentage of agarose and DNA either stuck in the well or prematurely ran out of the gel.

Also know, why are my PCR bands faint?

Popular Answers (1) First check your programming for each step of PCR cycle as the faint bands are due to several reasons like insufficient number of your cycles, low extension time, low annealing time, increased annealing temperature, decreased denaturing temperature, high or low denaturation time.

How many bands would appear on the electrophoresis gel?

The resulting gel will reveal two bands. Electrophoresis separates DNA fragments according to their relative size (molecular weight).


Moreaboutbiotechnology

Agarose gel electrophoresis is an easy way to separate DNA fragments by their sizes and visualize them. It is a common diagnostic procedure used in molecular biological labs.

Electrophoresis:

The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backbone. For this reason, when an electrical potential is placed on the DNA it will move toward the positive pole:

The rate at which the DNA will move toward the positive pole is slowed by making the DNA move through an agarose gel. This is a buffer solution (which maintains the proper pH and salt concentration) with 0.75% to 2.0% agarose added. The agarose forms a porous lattice in the buffer solution and the DNA must slip through the holes in the lattice in order to move toward the positive pole. This slows the molecule down. Larger molecules will be slowed down more than smaller molecules, since the smaller molecules can fit through the holes easier. As a result, a mixture of large and small fragments of DNA that has been run through an agarose gel will be separated by size. This is a graphic representation of an agarose gel made by "running" DNA molecular weight markers, an isolated plasmid, and the same plasmid after linearization with a restriction enzyme:

These gels are visualized on a U.V. trans-illuminator by staining the DNA with a fluorescent dye (ethidium bromide). The DNA molecular weight marker is a set of DNA fragments of known molecular sizes that are used as a standard to determine the sizes of your unknown fragments.

If you click on the figure you will see a short movie that simulates the movement of the DNA bands through the gel. When looking at the video, note that bands of a low molecular weight move very quickly through the gel while high molecular weight bands move very slowly.

Interpretation:

Much information can be derived from this gel. As you read the text below,

1.)  By looking at the migration of the DNA molecular weight standards, you can tell that the migration of DNA through an agarose gel is not linear with respect to size. If you graphed the distance traveled vs. the molecular weight of the fragment, you would see that there is a logarithmic relationship (i.e. small fragments travel much faster than large fragments).

2.) You can see that there is a big difference between the way a plasmid as isolated from the alkaline lysis prep will run vs. this same plasmid after it is cut with a restriction enzyme and linearized. This is because the plasmid will be found in many different supercoiled forms in the bacteria. When you isolate plasmid from a bacterial culture, you isolate all the different supercoiled forms of the plasmid, and each will migrate differently on the gel, giving you three major bands and many minor bands. When this mixture of supercoiled plasmids is cut with a restriction enzyme, the different forms linearize and unwind. As a result they all become identical and run at the same rate, and you see only one band on the gel.

3.) The molecular size of an unknown piece of DNA can be estimated by comparison of the distance that it travels with that of the molecular weight standards. This is only true for linear DNA. None of the supercoiled forms will migrate at a rate relative to linear DNA, which means that you can't use the DNA markers to estimate the molecular weight of a circular DNA molecule. To estimate the molecular weight of a plasmid, you must first linearize it. By looking at the gel above, the molecular size of the plasmid can be estimated at approximately 3.0 kilobases (kb). A more accurate estimate can be found by graphing the molecular weight of the standards (in base pairs) vs. the distance traveled on semi-log paper and using this graph to determine the molecular weight of the unknown. You will do this at the end of this experiment. Molecular size is the most important information derived from the agarose gel and the usual reason for running a gel.

In this experiment, you will linearize the plasmid that you isolated last week with a restriction enzyme. Then you will run this linearized plasmid on an agarose gel with the uncut version and a DNA marker to determine the size of your plasmid + insert, which will give you an estimate of the size of your insert.

Procedure:

1.) Put together the following reaction mixture for the restriction digestion:

ق.0 ul 10X Rest. Enzyme buffer

ك.0 ul plasmid DNA solution (from last week)

ـ.5 ul Restriction Enzyme (eg., HindIII)

Add the enzyme last, and always keep it on ice. The enzyme you will use will depend on the plasmid that you have, and will be told to you during class. 0.5 ul can't be measured with your pipetman. You must estimate it by the way it will look in the pipet tip (instruction will be given in class). Be sure to use a clean tip when taking the enzyme out of the tube. Put this reaction at 37oC for 45 minutes.

2.) When the digestion is complete, prepare to load the gel. In a new tube, place 17.0 ul of H2O and 3.0 ul of uncut plasmid DNA. Add 2.0 ul dye to each of the three sample tubes (DNA markers, uncut plasmid, and digested plasmid). Load 20.0 ul of DNA marker in to one well of the gel. Do this by sucking the solution into the pipet tip, placing the tip in the top of the well, and gently expelling the liquid into the well. The dye buffer in the DNA marker and samples contains glycerol which makes it more dense than H2O. This will cause the liquid to sink to the bottom of the well. Load 20.0 ul of the uncut plasmid and the restriction digestion.

3.) Turn on the power supply and electrophorese the samples at 110 V (Warning- be careful of the high voltage or you will be set down on your butt dramatically.) Electrophorese the samples until the dark blue dye is about 2 cm from the bottom of the gel

4.) Stain the gel by incubating it for 8 min in an ethidium bromide solution.

Ethidium bromide is very carcinogenic. Handle this gel only while wearing gloves. Never put unprotected fingers in the gel buffer solution.

5.) I will move the gel onto the U.V. trans illuminator and take a picture of it.

6.) I will denature the ethidium bromide by placing the gel in potassium permanganate solution for 5 minutes, then discard it.


Watch the video: Effect of DNA Supercoiling on Agarose Electrophoresis (September 2022).


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