DNA construct

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell.[1] These elements can be as small as a few thousand base pairs (kbp) of DNA carrying a single gene, or as large as hundreds of kbp for large-scale genomic studies. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. A DNA construct may express wildtype protein, prevent the expression of certain genes by expressing competitors or inhibitors, or express mutant proteins, such as deletion mutations or missense mutations. It can also prevent the expression of certain genes by encoding sequences of protein competitors or inhibitors. DNA constructs are widely adapted in molecular biology research for techniques such as DNA sequencing, protein expression, and RNA studies.

Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker.[2] Certain vectors can carry additional regulatory elements based on the expression system involved.

History

The first standardized vector, pBR220, was designed in 1977 by researchers in Herbert Boyer’s lab. The plasmid contains various restriction enzyme sites and a stable antibiotic-resistance gene free from transposon activities.[3]

In 1982, Jeffrey Vieira and Joachim Messing described the development of M13mp7-derived pUC vectors that consist of a multiple cloning site and allow for more efficient sequencing and cloning using a set of universal M13 primers. Three years later, the currently popular pUC19 plasmid was engineered by the same scientists.[4]

Modes of delivery

There are three general categories of DNA construct delivery: physical, chemical, and viral.[5] Physical methods, which deliver the DNA by physically penetrating the cell, include microinjection, electroporation, and biolistics.[6] Chemical methods rely on chemical reactions to deliver the DNA and include transformation with cells made competent using calcium phosphate as well as delivery via lipid nanoparticles.[7][8] Viral methods use a variety of viral vectors to deliver the DNA, including adenovirus, lentivirus, and herpes simplex virus[9]

Types of DNA constructs
A commonly used plasmid vector, pET28a[10]
  • Artificial chromosomes: commonly used in genome project studies due to its ability to hold inserts up to 350 kbp. These vectors are derived from the F plasmid, taking advantage of the high stability and conjugational ability introduced by the F factor.[11]
  • Bacteriophage Vectors are insertions carried by the bacteriophage λ genome can accommodate up to 12 kbp without disrupting the phage envelope. These vectors allow for efficient cloning as the phage can replicate within E. coli.
  • Fosmids are a hybrid between bacterial F plasmids and λ phage cloning techniques. Inserts are pre-packaged into phage particles, then inserted into the host cell with the ability to hold ~45 kbp. They are typically used to generate a DNA library due to their increased stability.[12]
  • Bacterial plasmids are vectors capable of holding inserts up to approximately 20 kbp in length. These types of constructs typically contain a gene offering antibiotic-resistance, an origin of replication, regulatory elements such as Lac inhibitors, a polylinker, and a protein tag which facilitates protein purification.[13]

See also

References
  1. Pinkert, Carl (2014). Transgenic animal technology: A laboratory handbook. Amsterdam: Elsevier. p. 692. ISBN 9780124095366.
  2. Carter, Matt; Shieh, Jennifer C. (2010), "Molecular Cloning and Recombinant DNA Technology", Guide to Research Techniques in Neuroscience, Elsevier, pp. 207–227, doi:10.1016/b978-0-12-374849-2.00009-4, ISBN 978-0-12-374849-2, retrieved 2020-10-24
  3. Bolivar, Francisco; Rodriguez, Raymond L.; Betlach, Mary C.; Boyer, Herbert W. (1977-11-01). "Construction and characterization of new cloning vehicles I. Ampicillin-resistant derivatives of the plasmid pMB9". Gene. 2 (2): 75–93. doi:10.1016/0378-1119(77)90074-9. ISSN 0378-1119.
  4. Yanisch-Perron, Celeste; Vieira, Jeffrey; Messing, Joachim (1985-01-01). "Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors". Gene. 33 (1): 103–119. doi:10.1016/0378-1119(85)90120-9. ISSN 0378-1119.
  5. Carter, Matt; Shieh, Jennifer C. (2010), "Gene Delivery Strategies", Guide to Research Techniques in Neuroscience, Elsevier, pp. 229–242, doi:10.1016/b978-0-12-374849-2.00010-0, ISBN 978-0-12-374849-2, retrieved 2020-10-24
  6. Mehierhumbert, S; Guy, R (2005-04-05). "Physical methods for gene transfer: Improving the kinetics of gene delivery into cells". Advanced Drug Delivery Reviews. 57 (5): 733–753. doi:10.1016/j.addr.2004.12.007.
  7. Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. (1987-11-01). "Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure". Proceedings of the National Academy of Sciences. 84 (21): 7413–7417. doi:10.1073/pnas.84.21.7413. ISSN 0027-8424. PMC 299306. PMID 2823261.
  8. Kingston, Robert E.; Chen, Claudia A.; Rose, John K. (2003). "Calcium Phosphate Transfection". Current Protocols in Molecular Biology. 63 (1): 9.1.1–9.1.11. doi:10.1002/0471142727.mb0901s63. ISSN 1934-3647.
  9. Robbins, Paul D.; Ghivizzani, Steven C. (1998). "Viral Vectors for Gene Therapy". Pharmacology & Therapeutics. 80 (1): 35–47. doi:10.1016/S0163-7258(98)00020-5.
  10. Shen, Aimee; Lupardus, Patrick J.; Morell, Montse; Ponder, Elizabeth L.; Sadaghiani, A. Masoud; Garcia, K. Christopher; Bogyo, Matthew (2009-12-02). Xu, Wenqing (ed.). "Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag". PLoS ONE. 4 (12): e8119. doi:10.1371/journal.pone.0008119. ISSN 1932-6203. PMC 2780291. PMID 19956581.
  11. Godiska, R.; Wu, C. -C.; Mead, D. A. (2013-01-01), Maloy, Stanley; Hughes, Kelly (eds.), "Genomic Libraries", Brenner's Encyclopedia of Genetics (Second Edition), San Diego: Academic Press, pp. 306–309, doi:10.1016/b978-0-12-374984-0.00641-0, ISBN 978-0-08-096156-9, retrieved 2020-11-06
  12. Hu, Bo; Khara, Pratick; Christie, Peter J. (2019-07-09). "Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli". Proceedings of the National Academy of Sciences. 116 (28): 14222–14227. doi:10.1073/pnas.1904428116. ISSN 0027-8424. PMC 6628675. PMID 31239340.
  13. Griffiths, Anthony J.F. (2015). Introduction To Genetic Analysis. New York: W.H. Freeman & Company. ISBN 978-1464188046.


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