Editing Genetic engineering (section)

=== Gene isolation and cloning ===
{{Main|Molecular cloning}}
The next step is to isolate the candidate gene. The [[Cell (biology)|cell]] containing the gene is opened and the DNA is purified.<ref>{{Cite book|url=https://books.google.com/books?id=g1v6WMHVkTgC|title=An Introduction to Genetic Engineering|last=Nicholl|first=Desmond S.T.|date=2008-05-29|publisher=Cambridge University Press|isbn=978-1-139-47178-7|page=34|language=en}}</ref> The gene is separated by using [[restriction enzymes]] to cut the DNA into fragments<ref>{{cite book |vauthors=Alberts B, Johnson A, Lewis J, etal |chapter= Isolating, Cloning, and Sequencing DNA |title=Molecular Biology of the Cell |edition= 4th |location=New York|publisher=Garland Science|year= 2002 |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK26837/ |isbn=0-8153-3218-1}}</ref> or [[polymerase chain reaction]] (PCR) to amplify up the gene segment.<ref>{{cite journal | vauthors = Kaufman RI, Nixon BT | title = Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria | journal = Journal of Bacteriology | volume = 178 | issue = 13 | pages = 3967–70 | date = July 1996 | pmid = 8682806 | pmc = 232662 | doi = 10.1128/jb.178.13.3967-3970.1996 }}</ref> These segments can then be extracted through [[gel electrophoresis]]. If the chosen gene or the donor organism's [[genome]] has been well studied it may already be accessible from a [[Library (biology)|genetic library]]. If the [[DNA sequence]] is known, but no copies of the gene are available, it can also be [[Gene synthesis|artificially synthesised]].<ref>{{cite journal | vauthors = Liang J, Luo Y, Zhao H | title = Synthetic biology: putting synthesis into biology | journal = Wiley Interdisciplinary Reviews: Systems Biology and Medicine | volume = 3 | issue = 1 | pages = 7–20 | year = 2011 | pmid = 21064036 | pmc = 3057768 | doi = 10.1002/wsbm.104 }}</ref> Once isolated the gene is [[Ligation (molecular biology)|ligated]] into a [[plasmid]] that is then inserted into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited copies of the gene are available.<ref>{{Cite web|url=http://www.fao.org/docrep/006/y4955e/y4955e06.htm|title=5. The Process of Genetic Modification|website=www.fao.org|access-date=2017-04-29}}</ref> The [[RK2 plasmid]] is notable for its ability to replicate in a wide variety of [[single-celled organism]]s, which makes it suitable as a genetic engineering tool.<ref>J M Blatny, T Brautaset, C H Winther-Larsen, K Haugan and S Valla: "Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon", ''Appl. Environ. Microbiol.'' 1997, Volume 63, Issue 2, p. 370</ref>

Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a [[Promoter (biology)|promoter]] and [[Terminator (genetics)|terminator]] region, which initiate and end [[Transcription (genetics)|transcription]]. A [[selectable marker]] gene is added, which in most cases confers [[antibiotic resistance]], so researchers can easily determine which cells have been successfully transformed. The gene can also be modified at this stage for better expression or effectiveness. These manipulations are carried out using [[recombinant DNA]] techniques, such as [[restriction digest]]s, ligations and molecular cloning.<ref>{{cite journal | vauthors = Berg P, Mertz JE | title = Personal reflections on the origins and emergence of recombinant DNA technology | journal = Genetics | volume = 184 | issue = 1 | pages = 9–17 | date = January 2010 | pmid = 20061565 | pmc = 2815933 | doi = 10.1534/genetics.109.112144 }}</ref>