Editing Molecular biology (section)

===Molecular cloning===
{{main|Molecular cloning}}
[[File:Transduction image.pdf|thumb|Transduction image]]
Molecular cloning is used to isolate and then transfer a DNA sequence of interest into a plasmid vector.<ref>{{Cite web|title=Foundations of Molecular Cloning - Past, Present and Future {{!}} NEB|url=https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future|access-date=2021-11-25|website=www.neb.com}}</ref> This recombinant DNA technology was first developed in the 1960s.<ref>{{Cite web|title=Foundations of Molecular Cloning - Past, Present and Future {{!}} NEB|url=https://www.neb.com/tools-and-resources/feature-articles/foundations-of-molecular-cloning-past-present-and-future|access-date=2021-11-04|website=www.neb.com}}</ref> In this technique, a [[DNA]] sequence coding for a protein of interest is [[clone (genetics)|cloned]] using [[polymerase chain reaction]] (PCR), and/or [[restriction enzyme]]s, into a [[plasmid]] ([[expression vector]]). The plasmid vector usually has at least 3 distinctive features: an origin of replication, a [[multiple cloning site]] (MCS), and a selective marker (usually [[antibiotic resistance]]). Additionally, upstream of the MCS are the [[promoter region]]s and the [[Transcription (genetics)|transcription]] start site, which regulate the expression of cloned gene.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by [[transformation (genetics)|transformation]] via uptake of naked DNA, [[bacterial conjugation|conjugation]] via cell-cell contact or by [[transduction (genetics)|transduction]] via viral vector. Introducing DNA into [[Eukaryote|eukaryotic]] cells, such as animal cells, by physical or chemical means is called [[transfection]]. Several different transfection techniques are available, such as calcium phosphate transfection, [[electroporation]], [[microinjection]] and [[liposome transfection]]. The plasmid may be integrated into the [[genome]], resulting in a stable transfection, or may remain independent of the genome and expressed temporarily, called a transient transfection.<ref name="cell">{{cite book|last1=Alberts|first1=Bruce|last2=Johnson|first2=Alexander|last3=Lewis|first3=Julian|last4=Raff|first4=Martin|last5=Roberts|first5=Keith|last6=Walter|first6=Peter | name-list-style = vanc |title=Isolating, Cloning, and Sequencing DNA|url=https://www.ncbi.nlm.nih.gov/books/NBK26837/|access-date=31 December 2016|language=en}}</ref><ref>{{cite book|last1=Lessard|first1=Juliane C.|title=Laboratory Methods in Enzymology: DNA|chapter=Molecular cloning|date=1 January 2013|volume=529|pages=85–98|doi=10.1016/B978-0-12-418687-3.00007-0|pmid=24011038|issn=1557-7988|isbn=978-0-12-418687-3}}</ref>

DNA coding for a protein of interest is now inside a cell, and the [[protein]] can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its [[tertiary structure]] can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.<ref>{{cite book|last1=Kokate |first1=Chandrakant|last2=Jalalpure|first2=Sunil S.|last3=Hurakadle|first3=Pramod J.| name-list-style = vanc |title=Textbook of Pharmaceutical Biotechnology|department=Expression Cloning|url=https://books.google.com/books?id=p70UCwAAQBAJ&pg=PA125|date=2016|publisher=Elsevier|page=125|access-date=2019-07-08|isbn=9788131239872}}</ref>