Editing Agarose gel electrophoresis (section)

==Migration of nucleic acids in agarose gel==
{{main|Gel electrophoresis of nucleic acids}}

===Factors affecting migration of nucleic acid in gel===
[[File:Plasmid miniprep.jpg|thumb|280px|Gels of plasmid preparations usually show a major band of supercoiled DNA with other fainter bands in the same lane. Note that by convention DNA gel is displayed with smaller DNA fragments nearer to the bottom of the gel. This is because historically DNA gels were run vertically and the smaller DNA fragments move downwards faster.]]

A number of factors can affect the migration of nucleic acids: the dimension of the gel pores (gel concentration), size of DNA being electrophoresed, the voltage used, the ionic strength of the buffer, and the concentration of intercalating dye such as ethidium bromide if used during electrophoresis.<ref>{{cite book |author1=G. Lucotte |author2=F. Baneyx |title= Introduction to Molecular Cloning Techniques |page=32 | year=1993 |publisher=Wiley-Blackwell |isbn=978-0471188490 }}</ref>

Smaller molecules travel faster than larger molecules in gel, and double-stranded DNA moves at a rate that is inversely proportional to the logarithm of the number of base pairs. This relationship however breaks down with very large DNA fragments, and separation of very large DNA fragments requires the use of [[pulsed field gel electrophoresis]] (PFGE), which applies alternating current from different directions and the large DNA fragments are separated as they reorient themselves with the changing field.<ref name=":0">{{cite journal | vauthors = Lee PY, Costumbrado J, Hsu CY, Kim YH | title = Agarose gel electrophoresis for the separation of DNA fragments | journal = Journal of Visualized Experiments | issue = 62 | date = April 2012 | pmid = 22546956 | pmc = 4846332 | doi = 10.3791/3923 }}</ref>

For standard agarose gel electrophoresis, larger molecules are resolved better using a low concentration gel while smaller molecules separate better at high concentration gel. Higher concentration gels, however, require longer run times (sometimes days).

The movement of the DNA may be affected by the [[Chemical structure|conformation]] of the DNA molecule, for example, [[DNA supercoil|supercoiled DNA]] usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present.<ref>{{cite book |url=https://books.google.com/books?id=Q6Yd-qYvx9UC&pg=PA97 |title=DNA Structure and Function |author = Richard R. Sinden |page= 97 |publisher=Academic Press Inc. |isbn=978-0126457506|date=1994-11-24 }}</ref> Gel electrophoresis of the plasmids would normally show the negatively supercoiled form as the main band, while nicked DNA (open circular form) and the relaxed closed circular form appears as minor bands. The rate at which the various forms move however can change using different electrophoresis conditions,<ref name=sambrook1>{{cite book |title=Molecular Cloning - A Laboratory Manual |author1=Joseph Sambrook |author2=David Russell |volume=1 |edition=3rd |chapter=Chapter 5, protocol 1 |page=5.5–5.6 |isbn=978-0-87969-577-4 }}</ref> and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.<ref>{{cite journal | vauthors = Aaij C, Borst P | title = The gel electrophoresis of DNA | journal = Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis | volume = 269 | issue = 2 | pages = 192–200 | date = May 1972 | pmid = 5063906 | doi = 10.1016/0005-2787(72)90426-1 }}</ref>

Ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence in gel during electrophoresis can affect its movement. For example, the positive charge of ethidium bromide can reduce the DNA movement by 15%.<ref name=":0" /> Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology.<ref>{{cite book |title=Biochemistry |author1=Donald Voet |author2=Judith G. Voet |edition=2nd |publisher=John Wiley & Sons |pages=[https://archive.org/details/biochemistry00voet_0/page/877 877–878] |year=1995 |isbn=978-0471586517 |url=https://archive.org/details/biochemistry00voet_0/page/877 }}</ref>

DNA damage due to increased [[cross-link]]ing will also reduce electrophoretic DNA migration in a dose-dependent way.<ref>{{cite journal | vauthors = Blasiak J, Trzeciak A, Malecka-Panas E, Drzewoski J, Wojewódzka M | title = In vitro genotoxicity of ethanol and acetaldehyde in human lymphocytes and the gastrointestinal tract mucosa cells | journal = Toxicology in Vitro | volume = 14 | issue = 4 | pages = 287–95 | date = August 2000 | pmid = 10906435 | doi = 10.1016/S0887-2333(00)00022-9 | bibcode = 2000ToxVi..14..287B }}</ref><ref>{{cite journal | vauthors = Lu Y, Morimoto K | title = Is habitual alcohol drinking associated with reduced electrophoretic DNA migration in peripheral blood leukocytes from ALDH2-deficient male Japanese? | journal = Mutagenesis | volume = 24 | issue = 4 | pages = 303–8 | date = July 2009 | pmid = 19286920 | doi = 10.1093/mutage/gep008 | doi-access = free }}</ref>

The rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. The resolution of large DNA fragments however is lower at high voltage. The mobility of DNA may also change in an unsteady field – in a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency.<ref name="zimm">{{cite journal | vauthors = Zimm BH, Levene SD | title = Problems and prospects in the theory of gel electrophoresis of DNA | journal = Quarterly Reviews of Biophysics | volume = 25 | issue = 2 | pages = 171–204 | date = May 1992 | pmid = 1518924 | doi = 10.1017/s0033583500004662 | s2cid = 27976751 | url = http://www.damtp.cam.ac.uk/user/gold/pdfs/teaching/ufk_papers/electrokinetics/zimm.pdf }}</ref>  This phenomenon can result in band inversion in field inversion gel electrophoresis (FIGE), whereby larger DNA fragments move faster than smaller ones.

===Migration anomalies===
* "Smiley" gels - this edge effect is caused when the voltage applied is too high for the gel concentration used.<ref>{{cite book |author1=G. Lucotte |author2=F. Baneyx |title= Introduction to Molecular Cloning Techniques |page=41 | year=1993 |publisher=Wiley-Blackwell |isbn=978-0471188490 }}</ref> 
* Overloading of DNA - overloading of DNA slows down the migration of DNA fragments.
* Contamination - presence of impurities, such as salts or proteins can affect the movement of the DNA.

===Mechanism of migration and separation===

The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis.<ref name="zimm"/><ref name="old & primrose">{{cite book |title=Principle of Gene Manipulation - An Introduction to Genetic Engineering |author1=Robert W. Old |author2=Sandy B. Primrose |page=[https://archive.org/details/principlesofgene00oldr/page/9 9] |publisher=Blackwell Scientific |edition=5th |isbn=9780632037124 |url=https://archive.org/details/principlesofgene00oldr/page/9 |date=1994-09-27 }}</ref>  The gel matrix is therefore responsible for the separation of DNA by size during electrophoresis, and a number of models exist to explain the mechanism of separation of biomolecules in gel matrix. A widely accepted one is the Ogston model which treats the polymer matrix as a sieve. A globular protein or a [[random coil]] DNA moves through the interconnected pores, and the movement of larger molecules is more likely to be impeded and slowed down by collisions with the gel matrix, and the molecules of different sizes can therefore be separated in this sieving process.<ref name="zimm" />

The Ogston model however breaks down for large molecules whereby the pores are significantly smaller than size of the molecule. For DNA molecules of size greater than 1 kb, a [[reptation]] model (or its variants) is most commonly used. This model assumes that the DNA can crawl in a "snake-like" fashion (hence "reptation") through the pores as an elongated molecule. A biased reptation model applies at higher electric field strength, whereby the leading end of the molecule become strongly biased in the forward direction and pulls the rest of the molecule along.<ref name="microfluidics">{{cite book |chapter-url=https://books.google.com/books?id=8wyBp-vKbdcC&pg=PA125 |title=Microfluidics for Biological Applications |editor1=Tian, Wei-Cheng |editor2=Finehout, Erin |chapter=Chapter 4 - Genetic Analysis in Miniaturized Electrophoresis Systems |author1=Li Zhu |author2=Hong Wang |page=125 |publisher=Springer |isbn=978-0-387-09480-9 |date=2009-03-02 }}</ref>  Real-time fluorescence microscopy of stained molecules, however, showed more subtle dynamics during electrophoresis, with the DNA showing considerable elasticity as it alternately stretching in the direction of the applied field and then contracting into a ball, or becoming hooked into a U-shape when it gets caught on the polymer fibres.<ref>{{cite journal | vauthors = Smith SB, Aldridge PK, Callis JB | title = Observation of individual DNA molecules undergoing gel electrophoresis | journal = Science | volume = 243 | issue = 4888 | pages = 203–6 | date = January 1989 | pmid = 2911733 | doi = 10.1126/science.2911733 | bibcode = 1989Sci...243..203S }}</ref><ref>{{cite journal | vauthors = Schwartz DC, Koval M | title = Conformational dynamics of individual DNA molecules during gel electrophoresis | journal = Nature | volume = 338 | issue = 6215 | pages = 520–2 | date = April 1989 | pmid = 2927511 | doi = 10.1038/338520a0 | bibcode = 1989Natur.338..520S | s2cid = 4249063 }}</ref>