what causes the DNA fragments to move through the gel

Simple laws of physics dictate that when current is applied to a medium containing charged species, those species volition migrate towards the opposite charge. Depending on the medium through which they are moving, other characteristics – such as the size of the species present – can touch on their move, leading to separation. This is the basis on which electrophoresis techniques, such every bit agarose gel electrophoresis, are built – techniques that are widely used across the life sciences.

In this article, nosotros volition consider how agarose gel electrophoresis works, how it can be interpreted and some of its purposes.

What is electrophoresis?

Electrophoresis is a technique that uses electrical current to divide DNA, RNA or proteins based on their physical properties such as size and charge.

What is agarose gel electrophoresis?

Agarose gel electrophoresis is a form of electrophoresis used for the separation of nucleic acid (DNA or RNA) fragments based on their size. Negatively charged Deoxyribonucleic acid/RNA migrates through the pores of an agarose gel towards the positively charged finish of the gel when an electric current is practical, with smaller fragments migrating faster. The resulting bands can and then be visualized using ultraviolet (UV) light.

RNA,¹ however, tends to form secondary structures – sometimes multiple unlike species for the aforementioned fragment – that bear on the way it migrates. Consequently, observed bands do not e'er correspond their truthful sizes and images are blurry. Native agarose gels (where conditions do non disrupt the natural structures of analytes) therefore tend non to be used for the analysis of RNA sizes, although they tin give an estimate of quantity and integrity. Alternatives include northern blotting and denaturing agarose gel electrophoresis^two .

Agarose gels may besides exist used to dissever proteins ^three based on their size and charge (unlike Deoxyribonucleic acid/RNA, proteins vary in accuse according to the amino acids incorporated). Still, due to the big pore sizes in agarose gels, proteins are often separated on polyacrylamide gels that accept smaller pores instead, offering greater resolution for small protein molecules.

We volition therefore focus on Dna agarose gel electrophoresis for the balance of this commodity.

How does gel electrophoresis piece of work?

Agarose is a component of agar. Information technology forms a 3D gel matrix of helical agarose molecules in supercoiled bundles held by hydrogen bonds, with channels and pores through which molecules are able to laissez passer. When heated, these hydrogen bonds break, turning the agarose to liquid and allowing information technology to be poured into a mold earlier it resets (Figure i).

Figure 1: Pore germination and temperature-induced state transition in agarose gel.

The percentage of agarose included in a gel impacts the pore sizes and thus the size of molecules that may pass through and speed at which they practise so. The higher the percentage of agarose, the smaller the pore size, thus the smaller the molecules able to laissez passer and the slower the migration. In the molecular biology lab, 0.vii-1% agarose gel is typically used for day-to-twenty-four hours DNA separations, offering good, clear differentiation of fragments in the range of 0.2-x kb. Larger fragments may be resolved using lower percentage gels, just they become very frail and hard to handle, while higher per centum gels volition give meliorate resolution of pocket-sized fragments but are brittle and may set unevenly.

As Dna is not visible to the naked eye, an intercalating dye such every bit ethidium bromide (EtBr) is incorporated in the gel during setting. This binds the Dna and fluoresces under UV low-cal, allowing the Dna fragments to be visualized. The more DNA present, the brighter the band.

Samples mixed with a loading dye are placed in one finish of the gel which is immersed in running buffer. An electrical current is then passed through the gel by electrodes at each end of the gel tank (Figure 2).

Effigy 2: Illustration of an agarose gel electrophoresis setup. The agarose gel sits in a tank of buffer, the samples mixed with loading dye are placed in wells at 1 finish of the gel and an electrical current is applied causing the negatively charged DNA to move towards the positive electrode (cathode).

When the samples have run far enough to obtain sufficient separation, the gel is removed from the tank and placed on a UV light box. The intercalating dye and so allows the sample bands to be visualized and their size determined in comparing to a DNA ladder with known ring sizes. The human relationship between migration distance and fragment size in non-linear, adding to the importance of including size markers as a guide (Figure 3).

Figure three: A) The illustration above depicts a typical result of Deoxyribonucleic acid electrophoresis. On the left, there is a size marker that is used as a reference for the length of the sample DNA fragments (in base pairs). To the right of the marking are three samples: Sample A, Sample B and Sample C. The epitome shows how smaller Deoxyribonucleic acid fragments move further through the agarose gel than the larger fragments of Dna. B) The graph to the right of the image shows the nonlinear, relationship between the size of the Deoxyribonucleic acid fragments and the distance migrated. Information technology is a negative bend, and equally DNA fragments get larger, they migrate less distance through the gel. Credit: Mckenzielower, reproduced under the Artistic Commons Attribution-Share Akin 4.0 International license.

Deoxyribonucleic acid gel electrophoresis steps, the gel electrophoresis machine, electrophoresis buffer and electrical separation

There are a number of central steps ⁴ involved in choosing, setting up, running and analyzing agarose gels that we will now consider.

ane. Determine the required gel percentage – 0.vii – one% agarose gel is typically acceptable for almost applications, only it is important to choose a percentage appropriate for your samples and expected fragment sizes. Combine the agarose powder with the same buffer type to be used to run the gel and rut to melt the mixture, avoiding boiling. Tris-acetate-ethylenediaminetetraacetic acrid (EDTA) (TAE) or tris-borate-EDTA (TBE) ⁵ are often the buffers of choice, equally tris-acrid solutions are effective buffers for slightly basic conditions, keeping DNA deprotonated and soluble in h2o. The EDTA, a chelating agent, inactivates nucleases that may damage the Dna being analyzed.

2. Pour a gel – Choose a gel casting mold and rummage of the desired size, giving a sufficient number of wells for all samples and ladders and well chapters to concord the quantity of each sample to be loaded. Secure the open ends of the mold with a casting frame or tape to contain the gel while information technology sets. Add DNA intercalating dye into the bottom of the mold – for EtBr, 0.ii-0.5 µ thou/ml is typically used. Prove that EtBr is a mutagen is still currently debated but consequently, many labs have moved over to alternatives ⁶ such every bit GelRed. Add the gel, conscientious not to overfill the mold, and ensure the intercalating dye is evenly mixed. Don't pour the gel when it's too hot or the mold may warp or suspension.

three. Mix samples/ladders with loading dye – Loading dyes perform multiple functions. They permit the user to see where their otherwise colorless sample is, making it easier to pipette the sample into the well accurately and thus reducing the likelihood of cantankerous-contamination of samples betwixt the wells. When the gel is running, the dye migrates with the sample, assuasive the user to tell where in the gel the sample has reached and prevent it from running too far and being lost into the buffer. DNA samples without loading dye volition also tend to disperse into the running buffer when loaded equally they are less dense. Most loading dyes therefore contain glycerol or Ficoll which makes the sample-dye mix denser, and then it settles in the lesser of the wells. Bromophenol blue is a pop colorant pick, but some also contain additional dyes such equally xylene cyanol. While loading dyes can be purchased, many labs choose to make their own.⁷

If you are running very modest volumes of sample (east.one thousand., less than 5 µ l), it may be advantageous to add a little h2o at this stage to brand information technology easier to load the gel wells effectively and evenly. Every bit, if yous expect the concentration of Deoxyribonucleic acid in some samples to be much college than others, it may also be necessary to add together water to your sample-dye mix of these concentrated samples at this phase too. If you do not, the strong bespeak given by these bands during visualization can mask weaker bands or require the strong bands to be over exposed to view the weaker ones, creating vivid, distorted areas on the gel prototype.

four. Load the gel – Remove the casting frame/tape from the set gel and place it in the gel tank, ensuring that the wells are at the negative stop (black electrodes). Fill the tank with running buffer (TAE or TBE) then that the gel is submerged. Carefully remove the comb and gently pipette the sample-dye (and water if used) mix into the wells. Endeavor to avert touching the edges of the wells with the pipette tip as they may suspension and allow i sample to run into the next. Overloading wells can have the same event. Large amounts of DNA tin can besides slow down DNA migration during running. Load marking ladders, preferably one at each end of the sample row. Gels may not e'er run in a perfectly directly line and then having a ladder at each finish makes it easier to decide the sizes of fragments present. A diversity of ladders are bachelor with varying sizes indicated; chose 1 that is nearly appropriate for the fragment sizes you expect to run across.

5. Run the gel – Identify the hat on the tank with the electrodes black to black and cherry-red to cherry-red and plug the electrodes into a power pack, also black to black and red to red. This, along with the gel tank, makes up the gel electrophoresis machine. Brand certain that the electrodes and lid are the correct way effectually otherwise your samples will run backwards out of the wells and into the running buffer. Set the fourth dimension and voltage your gel volition be run at; 120 V for 35 mins is a practiced approximation, nonetheless this should exist tailored to the gel percentage used and expected fragment sizes beingness separated to give expert electrical separation. Applying current to an agarose gel volition cause information technology to heat upward, the higher the voltage the more information technology will heat so when running low percentage gels, information technology is advisable to utilise lower voltages to forbid melting. Information technology can be tempting to increase the voltage to brand a gel run faster. However, this can result in "smiley gels", where the bands are curved upwards at each cease, making it hard to determine the right band size. This is where the gel has started to cook slightly making the bands run unevenly. This tin can too cause bands to announced smeary and poorly defined.

six. Visualization – Once the samples have run most of the manner down the gel (the dye forepart will make this visible), plow off the power pack. Wearing gloves, gently remove the gel in the mold from the tank, draining off excess running buffer, and transfer to a UV box in an appropriate container for visualization. Change gloves to prevent contamination of surrounding surfaces, door handles etc. with intercalating dye from the gel or running buffer. If Dna fragments are required for downstream applications, the corresponding bands tin be advisedly excised from the gel with a scalpel blade while placed on a UV lite box in a dark room. Ensure y'all wear a UV confront shield and go along skin covered while the light box is on to prevent harm to your skin or optics by the UV light.

How to read gel electrophoresis

Agarose gels may be visualized on a UV light box in a dark room or using a cocky-independent light box linked to a camera. Whichever system is utilized, UV light is shone through the gel from below and bands of Deoxyribonucleic acid fluoresce thank you to the intercalating dye spring to them. This may be captured using a photographic camera with a specialized UV filter for your records. Marker ladders come up with a guide to betoken the size of each band they include. By comparing this to bands in sample lanes, the sizes of the bands can therefore exist determined. The relative amount of DNA between samples may too be compared, as college DNA concentrations will produce brighter bands. An case is shown in Figure 4.

Figure 4: Agarose gel (ii%) assay of PCR-amplified products from Dna extracted from a bronchoalveolar lavage (BAL) diagnostic specimen of a patient with pulmonary symptoms. Credit: The Centers for Disease Control and Prevention.

What is the purpose of gel electrophoresis?

There are a number of reasons why the separation of DNA fragments may be desirable, many of which are widely applicable beyond the life science disciplines. Let'south consider some mutual purposes.

• Visualization of sample Deoxyribonucleic acid
  Separating and visualizing Deoxyribonucleic acid fragments allows a user to determine:
  
  Whether Deoxyribonucleic acid is present in a sample – This can confirm, for instance, if a DNA extraction or a PCR has worked and in the context of a diagnostic exam tin therefore determine if a sample is positive or negative.
  
  The sizes of DNA fragments present – If performing a PCR or brake digest, for example, is the band of the expected size? This can confirm if genetic engineering experiments have been successful or may indicate the presence or absence of genetic insertions, deletions ⁸ or repeat regions ⁹ that tin can exist used as a diagnostic tool for some genetic conditions. When preparing Deoxyribonucleic acid for next-generation sequencing, information technology is important that the fragmented Dna for library preparation is of the right size for efficient sequencing.
  
  The amount of DNA nowadays – Although there are more accurate methods for precise DNA quantification,^ten such as ultraviolet-visible spectroscopy, when running samples on a gel, the intensity of the band produced tin give a rough idea of the amount of Deoxyribonucleic acid in a sample relative to other samples.
  
  How clean the sample is – While a smeared band may be expected in some contexts, such as when running whole genomic DNA, a articulate, well-baked ring may generally exist expected from a PCR or restriction digestion. Diffuse bands or smears may point suboptimal PCR conditions or primers, incomplete digestion or the presence of interfering contaminants such as RNA in the case of a DNA sample.
  
• Separation of DNA fragments for purification
  Where DNA fragments are required for downstream applications, such as cloning,¹¹ or following restriction digestion, information technology may be desirable to separate out DNA fragments of a specific size from others in the total sample. To attain this, the digested or amplified sample may exist run out on a gel and the piece of gel containing the fragments of interest excised. Clean-upwards kits and protocols ^{12 , 13}  are available to purify the DNA from the agarose gel before proceeding to downstream steps.
  
• Separation of Dna fragments for Southern blotting
  Southern blotting is a technique used to detect specific Dna sequences in a sample. In social club to do that though, the DNA fragments kickoff accept to exist separated by agarose gel electrophoresis before they can be probed for target sequences.
  
• Electrophoretic mobility shift assays (EMSAs)
  EMSA'south,^fourteen besides called gel shift assays, are used to find interactions between proteins and nucleic acids. Examples might include the binding of transcription factors ^xv that promote or forestall gene expression. When a protein binds to a fragment of Dna, it will alter the way it migrates through an agarose gel, producing a "shift". Therefore, past running different combinations of Deoxyribonucleic acid fragments with and without a putative Dna binding protein, it is possible to determine when binding has or has non occurred and thus determine the target sequence.

Deoxyribonucleic acid agarose gel electrophoresis glossary

Term	Definition
Agarose gel electrophoresis	A form of electrophoresis used for the separation of macromolecules, such equally Dna fragments, in an agarose matrix.
Chelating agent	A chemical chemical compound that reacts with metal ions to form stable, h2o-soluble metal complexes.
Denaturing agarose gels	Agarose gels run nether atmospheric condition that disrupt the natural construction of DNA, RNA or proteins, causing them to unfold.
Deprotonation	Removal of a proton (H+).
Dna ladder/mark ladder	A solution of DNA fragments of known sizes that tin can be used to extrapolate the sizes of fragments in unknown samples.
Electric separation	The separation of biomolecules, such as DNA, RNA or proteins, according to their size and/or charge using electrical current.
Electrophoresis	A technique that uses electrical current to separate Deoxyribonucleic acid, RNA or proteins based on their physical properties such equally size and charge.
Electrophoretic mobility shift assays (EMSA)	Too called gel shift assays, EMSAs are an electrophoresis-based technique used to detect interactions betwixt proteins and nucleic acids.
Gel electrophoresis automobile	Equipment used to perform gel electrophoresis that normally consists of a gel tank and power pack with connecting electrodes.
Intercalating dye	Dyes that demark double stranded Dna, enabling them to be visualized under UV calorie-free.
Loading dye	A dye used to fix DNA ladders and samples for electrophoresis that enables them to exist seen with the naked eye and increases their density to forestall dispersion.
Mutagen	A chemic or physical phenomenon that promotes errors in Deoxyribonucleic acid replication.
Native agarose gels	Agarose gels run under conditions that allow the preservation of the natural structure of biomolecules such as Deoxyribonucleic acid, RNA or proteins.
Northern blot	A technique used to observe specific RNA sequences in a sample that employs electrophoresis for sample separation.
Nucleases	Enzymes that cleave nucleic acids such as Deoxyribonucleic acid or RNA.
Polyacrylamide gel electrophoresis	A form of electrophoresis used for the separation of macromolecules, such as nucleic acids and proteins, in a polymerized acrylamide matrix.
Restriction digestion	A procedure that uses enzymes to cut DNA at specific sites according to the surrounding DNA sequence.
Running buffer	The buffer used to fill the tank in which the gel is immersed and will be run. It helps to maintain stable conditions.
Secondary structure	The 3D construction adopted past a polypeptide or polynucleotide resulting from electrostatic attractions betwixt neighboring residues.
Southern blot	A technique used to detect specific DNA sequences in a sample that employs electrophoresis for sample separation.
Transcription factors	Proteins involved in the process of converting, or transcribing, Deoxyribonucleic acid into RNA.

References

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two. Masek T, Vopalensky Five, Suchomelova P, Pospisek G. Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem. 2005;336(1):46-50. doi:10.1016/j.ab.2004.09.010

3. Krizek DM, Rick ME. Agarose gel electrophoresis of proteins. Curr Protoc Cell Biol. 2002;15(1):6.7.1-half dozen.seven.13. doi:10.1002/0471143030.cb0607s15

4. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of Dna fragments. J Vis Exp. 2012;(62):3923. doi:10.3791/3923

v. Sanderson BA, Araki N, Lilley JL, Guerrero G, Lewis LK. Modification of gel architecture and TBE/TAE buffer limerick to minimize heating during agarose gel electrophoresis. Anal Biochem. 2014;454:44-52. doi:ten.1016/j.ab.2014.03.003

half dozen. Hall AC. A comparison of Dna stains and staining methods for agarose gel electrophoresis. bioRxiv. 2019. doi:x.1101/568253

7. Deoxyribonucleic acid gel-loading dye (10X). Common cold Spring Harb Protoc. 2008;2008(8):pdb.rec11373. doi:x.1101/pdb.rec11373

8. Schwarz MJ. Deoxyribonucleic acid diagnosis of cystic fibrosis. Ann Clin Biochem. 1998;35(five):584-610. doi:10.1177/000456329803500502

ix. Marwal A, Sahu AK, Gaur RK. Chapter sixteen - Molecular Markers: Tool for Genetic Analysis. In: Verma Every bit, Singh A, eds. Animal Biotechnology. Academic Press; 2014:289-305. doi:10.1016/B978-0-12-416002-6.00016-X

10. Tweedie JW, Stowell KM. Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following handling with a restriction endonuclease or Deoxyribonucleic acid topoisomerase I. Biochem Mol Biol Educ. 2005;33(ane):28-33. doi:10.1002/bmb.2005.494033010410

eleven. Molnar C, Gair J. Chapter 10.1 Cloning and Genetic Engineering. In: Concepts of Biology. Published online May 14, 2015. Accessed February 2, 2022. https://opentextbc.ca/biology/chapter/x-1-cloning-and-genetic-engineering/

12. Balletbó A. DNA purification from an agarose gel (protocol for NucleoSpin® pCR clean-upward gel extraction kit). protocols.io. Published September 22, 2019. Accessed Feb ii, 2022. doi:10.17504/protocols.io.7hrhj56

13. Downey Northward. Extraction of DNA from Agarose Gels. In: Casali Northward, Preston A, eds. E. coli Plasmid Vectors: Methods and Applications. Methods in Molecular Biology^TM. Humana Printing; 2003:137-139. doi:10.1385/i-59259-409-iii:137

xiv. Hellman LM, Fried MG. Electrophoretic mobility shift analysis (EMSA) for detecting protein–nucleic acid interactions. Nat Protoc. 2007;2(8):1849-1861. doi:10.1038/nprot.2007.249

15. Yousaf N, Gould D. Demonstrating Interactions of Transcription Factors with Dna by Electrophoretic Mobility Shift Assay. In: Gould D, ed. Mammalian Synthetic Promoters. Methods in Molecular Biology. Springer; 2017:xi-21. doi:10.1007/978-1-4939-7223-4_2

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