Editing Agarose (section)

==Properties==
Agarose is available as a white powder which dissolves in near-boiling water, and forms a gel when it cools. Agarose exhibits the phenomenon of thermal [[hysteresis]] in its liquid-to-gel transition, i.e. it gels and melts at different temperatures. The gelling and melting temperatures vary depending on the type of agarose. Standard agaroses derived from ''[[Gelidium]]'' has a gelling temperature of {{convert|34|-|38|C}} and a melting temperature of {{convert|90|-|95|C}}, while those derived from ''[[Gracilaria]]'', due to its higher [[Methoxy group|methoxy]] substituents, has a gelling temperature of {{convert|40|-|52|C}} and melting temperature of {{convert|85|-|90|C}}.<ref>{{cite book |url= https://books.google.com/books?id=YHMrAAAAYAAJ&pg=PA25 |title=Workshop on Marine Algae Biotechnology: Summary Report |page=25 |publisher=National Academy Press |year=1986 }}</ref> The melting and gelling temperatures may be dependent on the concentration of the gel, particularly at low gel concentration of less than 1%. The gelling and melting temperatures are therefore given at a specified agarose concentration.

Natural agarose contains uncharged methyl groups and the extent of methylation is directly proportional to the gelling temperature. Synthetic methylation however have the reverse effect, whereby increased methylation lowers the gelling temperature.<ref name="lonza">{{cite web |url=http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Appendix_B_-_Agarose_Physical_Chemistry.pdf |archive-url=https://ghostarchive.org/archive/20221009/http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Appendix_B_-_Agarose_Physical_Chemistry.pdf |archive-date=2022-10-09 |url-status=live |title=Appendix B: Agarose Physical Chemistry |work=Lonza Group }}</ref> A variety of chemically modified agaroses with different melting and gelling temperatures are available through chemical modifications.

The agarose in the gel forms a meshwork that contains pores, and the size of the pores depends on the concentration of agarose added. On standing, the agarose gels are prone to [[Syneresis (chemistry)|syneresis]] (extrusion of water through the gel surface), but the process is slow enough to not interfere with the use of the gel.<ref>{{cite book |url= https://books.google.com/books?id=7ShrFsmKjT0C&pg=PA149|title=Functional Properties of Food Macromolecules | veditors = Hill SE, Ledward DA, Mitchell JR |page=149 |publisher=Springer |year=1998 |isbn=978-0-7514-0421-0 }}</ref><ref>{{cite book |url= https://books.google.com/books?id=l_OGMwaGkgQC&pg=PA102|title=Biodegradable Hydrogels for Drug Delivery | vauthors = Park H, Park K, Shalaby WS |page=102 |publisher=CRC Press |year=1993 |isbn=978-1566760041 }}</ref>

Agarose gel can have high gel strength at low concentration, making it suitable as an anti-convection medium for [[gel electrophoresis]]. Agarose gels as dilute as 0.15% can form slabs for gel electrophoresis.<ref name="serwer"/> The agarose polymer contains charged groups, in particular [[pyruvate]] and [[sulfate]].<ref name="lonza" /> These negatively charged groups can slow down the movement of DNA molecules in a process called [[electroendosmosis]] (EEO).

'''Low EEO (LE) agarose''' is therefore generally preferred for use in agarose [[gel electrophoresis of nucleic acids]]. Zero EEO agaroses are also available but these may be undesirable for some applications as they may be made by adding positively charged groups that can affect subsequent enzyme reactions.<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual | vauthors = Sambrook J, Russell D |volume=1 |edition=3rd |chapter=Chapter 5, protocol 1 |page=5.7 |isbn=978-0-87969-577-4 }}</ref> Electroendosmosis is a reason agarose is used preferentially over agar as agaropectin in agar contains a significant amount of negatively charged sulphate and carboxyl groups. The removal of agaropectin in agarose substantially reduces the EEO, as well as reducing the non-specific adsorption of biomolecules to the gel matrix. However, for some applications such as the electrophoresis of serum protein, a high EEO may be desirable, and agaropectin may be added in the gel used.<ref>{{cite book |url=https://books.google.com/books?id=z-bZ_FZHqRcC&pg=PA7 |title=Protein Electrophoresis in Clinical Diagnosis | vauthors = Keren D |pages=7–8 |publisher=CRC Press |date=26 September 2003|isbn=978-0340812136 }}</ref>

'''LE agarose''' is said to be better for preparative electrophoresis, i.e. when DNA needs to be extracted from an agarose gel.<ref>{{Cite web |last=Martin |first=Katherine |title=Agarose LE vs. Agarose – What’s the Difference? |url=https://goldbio.com/articles/article/Agarose-LE-vs-Agarose-the-Difference |access-date=2024-09-19 |website=Gold Biotechnology}}</ref>

===Low melting and gelling temperature agaroses===
The melting and gelling temperatures of agarose can be modified by chemical modifications, most commonly by hydroxyethylation, which reduces the number of intrastrand hydrogen bonds, resulting in lower melting and setting temperatures compared to standard agaroses.<ref>{{cite book |title=Molecular Cloning - A Laboratory Manual | vauthors = Maniatis T, Fritsch EF, Sambrook J |volume=1 |chapter=Chapter 5, protocol 6 |page=5.29 |isbn=978-0879695774 }}</ref>  The exact temperature is determined by the degree of substitution, and many available low-melting-point (LMP) agaroses can remain fluid at {{convert|30|-|35|C}} range. This property allows [[Enzyme|enzymatic]] manipulations to be carried out directly after the DNA gel electrophoresis by adding slices of melted gel containing DNA fragment of interest to a reaction mixture. The LMP agarose contains fewer of the sulphates that can affect some enzymatic reactions, and is therefore preferably used for some applications. 

Hydroxyethylated agarose also has a smaller pore size (~90 nm) than standard agaroses.<ref name="Griess Moreno Easom Serwer 1989 pp. 1475–1484">{{cite journal | last1=Griess | first1=Gary A. | last2=Moreno | first2=Elena T. | last3=Easom | first3=Richard A. | last4=Serwer | first4=Philip | title=The sieving of spheres during agarose gel electrophoresis: Quantitation and modeling | journal=Biopolymers | publisher=Wiley | volume=28 | issue=8 | year=1989 | issn=0006-3525 | doi=10.1002/bip.360280811 | pages=1475–1484}}</ref> Hydroxyethylation may reduce the pore size by reducing the packing density of the agarose bundles, therefore LMP gel can also have an effect on the time and separation during electrophoresis.<ref>{{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 | volume = 62 | issue = 62 | page = 3923 | date = April 2012 | pmid = 22546956 | pmc = 4846332 | doi = 10.3791/3923 }}</ref> Ultra-low melting or gelling temperature agaroses may gel only at {{convert|8|-|15|C}}.