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Molecular cloning

Diagram of molecular cloning using bacteria and plasmids

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.[1] The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.[2]

In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically modified microorganisms (GMOs).[3] This process takes advantage of the fact that a single bacterial cell can be induced to take up and replicate a single recombinant DNA molecule. This single cell can then be expanded exponentially to generate a large number of bacteria, each of which contains copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them. The idea arose that different DNA sequences could be inserted into a plasmid and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.[4]

Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. There is also a lower chance of success when inserting large-sized DNA sequences. Inserts larger than 10kbp have very limited success, but bacteriophages such as bacteriophage λ can be modified to successfully insert a sequence up to 40 kbp.[5]

  1. ^ Watson JD (2007). Recombinant DNA: genes and genomes: a short course. San Francisco: W.H. Freeman. ISBN 978-0-7167-2866-5.
  2. ^ Patten CL, Glick BR, Pasternak J (2009). Molecular Biotechnology: Principles and Applications of Recombinant DNA. Washington, D.C: ASM Press. ISBN 978-1-55581-498-4.
  3. ^ Brown T (2006). Gene cloning and DNA analysis: an introduction. Cambridge, MA: Blackwell Pub. ISBN 978-1-4051-1121-8.
  4. ^ Garrett RH, Grisham CM (2013). Biochemistry (Fifth ed.). Brooks/Cole, Cengage Learning. ISBN 978-1-133-10629-6. OCLC 777722371.
  5. ^ Garrett RH, Grisham CM (2010). Biochemistry (Fourth ed.). Belmont, CA, Brooks/Cole: Cengage Learning. p. 380. ISBN 978-0-495-10935-8. OCLC 297392560.

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