Insert (molecular biology)

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In Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism.[1]

Inserts can range from physical nucleotide additions using a technique system or the addition of artificial structures on a molecule via mutagenic chemicals, such as ethidium bromide or crystals.

Inserts into the genome of an organism normally occur due to natural causes. These causes include environmental conditions and intracellular processes. Environmental inserts range from exposure to radioactive radiation such as Ultraviolet, mutagenic chemicals, or DNA viruses. Intracellular inserts can occur through heritable changes in parent cells or errors in DNA replication or DNA repair.

Gene insertion techniques can be used for characteristic mutations in an organism for a desired phenotypic gene expression. A gene insert change can be expressed in a large variety of ends. These variants can range from the loss, or gain, of protein function to changes in physical structure i.e., hair, or eye, color. The goal of changes in expression are focused on a gain of function in proteins for regulation[2] or to termination of cellular function for prevention of disease.[3] The results of the variations are dependent on the place in the genome the addition, or mutation is located. The aim is to learn, understand, and possibly predict the expression of genetic material in organisms using physical and chemical analysis. To see the results of genetic mutations, or inserts, techniques such as DNA sequencing, gel electrophoresis, immunoassay, or microscopy  can observe mutation.

History[edit]

The field has expanded significantly since the publication in 1973 with biochemists Stanley N. Cohen and Herbert W. Boyer by using E. coli bacteria to learn how to cut fragments, rejoin different fragments, and insert the new genes.[4] The field has expanded tremendously in terms of precision and accuracy since then. Computers and technology have made it technologically easier to achieve narrowing of error and expand understanding in this field. Computers having a high capacity for data and calculations which made processing the large volume of information tangible, i.e., the use of ChIP and gene sequence.

Techniques and protocols[edit]

Homology directed repair (HDR) is a technique repairs breaks or lesions in DNA molecules. The most common technique to add inserts to desired sequences is the use of homologous recombination.[5] This technique has a specific requirement where the insert can only be added after it has been introduced to the nucleus of the cell, which can be added to the genome mostly during the G2 and S phases in the cell cycle.[6]

CRISPR gene editing[edit]

CRISPR gene editing based on Clustered regularly interspaced short palindromic repeats (CRISPR) -Cas9 is an enzyme that uses the gene sequences[7] to help control, cleave, and separate specific DNA sequences that are complementary to a CRISPR sequence.[8][9] These sequences and enzymes were originally derived from bacteriophages.[10] The importance of this technique in the field of genetic engineering is that it gives the ability to have highly precise targeted gene editing and the cost factor for this technique is low compared to other tools.[11][12][13] The ability to insert DNA sequences into the organism is easy and fast, although it can run into expression issues in higher complex organisms.[14][15]

Transcription activator-like effector nuclease[edit]

Transcription activator-like effector nuclease, TALENs, are a set of restriction enzymes that be created to cut out desired DNA sequences.[16] These enzymes are mostly used in combination with CRISPR-CAS9, Zinc finger nuclease, or HDR. The main reason for this is the ability for these enzymes to have the precision to cut and separate the desired sequence within a gene.

Zinc finger nuclease[edit]

Zinc finger nucleases are genetically engineered enzymes that combine fusing a zinc finger DNA-binding domain on a DNA-cleavage domain. These are also combined with CRISPR-CAS9 or TALENs to gain a sequence-specific addition, or deletion, within the genome of more complex cells and organisms.[17]

Gene gun[edit]

The gene gun, also known as a biolistic particle delivery system, is used to deliver transgenes, proteins, or RNA into the cell. It uses a micro-projectile delivery system that shoots coated particles of a typical heavy metal that has DNA of interest into cells using high speed. The genetic material will penetrate the cell and deliver the contents over a space area. The use of micro-projectile delivery systems is a technique known as biolistic.[18]

References[edit]

  1. ^ "insert - Terminology of Molecular Biology for insert – GenScript". www.genscript.com. Retrieved 22 October 2017.
  2. ^ Hahne JC, Lampis A, Valeri N (February 2021). "Vault RNAs: hidden gems in RNA and protein regulation". Cellular and Molecular Life Sciences. 78 (4): 1487–1499. doi:10.1007/s00018-020-03675-9. PMC 7904556. PMID 33063126.
  3. ^ Levine B, Kroemer G (January 2019). "Biological Functions of Autophagy Genes: A Disease Perspective". Cell. 176 (1–2): 11–42. doi:10.1016/j.cell.2018.09.048. PMC 6347410. PMID 30633901.
  4. ^ "Herbert W. Boyer and Stanley N. Cohen". Science History Institute. 2016-06-01. Retrieved 2021-04-19.
  5. ^ Malzahn A, Lowder L, Qi Y (2017-04-24). "Plant genome editing with TALEN and CRISPR". Cell & Bioscience. 7 (1): 21. doi:10.1186/s13578-017-0148-4. PMC 5404292. PMID 28451378.
  6. ^ Prill K, Dawson JF (2020). "Homology-Directed Repair in Zebrafish: Witchcraft and Wizardry?". Frontiers in Molecular Biosciences. 7: 595474. doi:10.3389/fmolb.2020.595474. PMC 7793982. PMID 33425990.
  7. ^ Mojica FJ, Rodriguez-Valera F (September 2016). "The discovery of CRISPR in archaea and bacteria". The FEBS Journal. 283 (17): 3162–9. doi:10.1111/febs.13766. hdl:10045/57676. PMID 27234458. S2CID 42827598.
  8. ^ Barrangou R (February 2015). "The roles of CRISPR-Cas systems in adaptive immunity and beyond". Current Opinion in Immunology. 32: 36–41. doi:10.1016/j.coi.2014.12.008. PMID 25574773.
  9. ^ Oh JH, van Pijkeren JP (2014-09-29). "CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri". Nucleic Acids Research. 42 (17): e131. doi:10.1093/nar/gku623. PMC 4176153. PMID 25074379.
  10. ^ Ishino Y, Krupovic M, Forterre P (April 2018). Margolin W (ed.). "History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology". Journal of Bacteriology. 200 (7): e00580–17, /jb/200/7/e00580–17.atom. doi:10.1128/JB.00580-17. PMC 5847661. PMID 29358495.
  11. ^ Ebrahimi V, Hashemi A (August 2020). "Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: A review". Gene. 753: 144813. doi:10.1016/j.gene.2020.144813. PMID 32470504.
  12. ^ Aird EJ, Lovendahl KN, St Martin A, Harris RS, Gordon WR (2018-05-31). "Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template". Communications Biology. 1 (1): 54. doi:10.1038/s42003-018-0054-2. PMC 6123678. PMID 30271937.
  13. ^ Maganti HB, Bailey AJ, Kirkham AM, Shorr R, Pineault N, Allan DS (March 2021). "Persistence of CRISPR/Cas9 gene edited hematopoietic stem cells following transplantation: A systematic review and meta-analysis of preclinical studies". Stem Cells Translational Medicine. 10 (7): 996–1007. doi:10.1002/sctm.20-0520. PMID 33666363.
  14. ^ Bi H, Fei Q, Li R, Liu B, Xia R, Char SN, et al. (July 2020). "Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production". Plant Biotechnology Journal. 18 (7): 1526–1536. doi:10.1111/pbi.13315. PMC 7292542. PMID 31821678.
  15. ^ Charpentier E, Marraffini LA (June 2014). "Harnessing CRISPR-Cas9 immunity for genetic engineering". Current Opinion in Microbiology. 19: 114–119. doi:10.1016/j.mib.2014.07.001. PMC 4155128. PMID 25048165.
  16. ^ Boch J (February 2011). "TALEs of genome targeting". Nature Biotechnology. 29 (2): 135–6. doi:10.1038/nbt.1767. PMID 21301438. S2CID 304571.
  17. ^ Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, et al. (July 2008). "Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification". Molecular Cell. 31 (2): 294–301. doi:10.1016/j.molcel.2008.06.016. PMC 2535758. PMID 18657511.
  18. ^ O'Brien JA, Lummis SC (June 2011). "Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles". BMC Biotechnology. 11 (1): 66. doi:10.1186/1472-6750-11-66. PMC 3144454. PMID 21663596.