DNA construct

Last updated

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] 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. This gene can be cloned from a naturally occurring gene, [2] or synthetically constructed. [3] The vector can be delivered using physical, chemical or viral methods. [4] 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. [5]

Contents

DNA constructs can be as small as a few thousand base pairs (kbp) of DNA carrying a single gene, using vectors such as plasmids or bacteriophages, or as large as hundreds of kbp for large-scale genomic studies using an artificial chromosome. [2] 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. DNA constructs are widely adapted in molecular biology research for techniques such as DNA sequencing, protein expression, and RNA studies. [5]

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. [6]

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. [7]

Construction

The gene on a DNA sequence of interest can either be cloned from an existing sequence or developed synthetically. To clone a naturally occurring sequence in an organism, the organism's DNA is first cut with restriction enzymes, which recognize DNA sequences and cut them, around the target gene. The gene can then be amplified using polymerase chain reaction (PCR). Typically, this process includes using short sequences known as primers to initially hybridize to the target sequence; in addition, point mutations can be introduced in the primer sequences and then copied in each cycle in order to modify the target sequence. [2]

It is also possible to synthesize a target DNA strand for a DNA construct. Short strands of DNA known as oligonucleotides can be developed using column-based synthesis, in which bases are added one at a time to a strand of DNA attached to a solid phase. Each base has a protecting group to prevent linkage that is not removed until the next base is ready to be added, ensuring that they are linked in the correct sequence. Oligonucleotides can also be synthesized on a microarray, which allows for tens of thousands of sequences to be synthesized at once, in order to reduce cost. [3] To synthesize a larger gene, oligonucleotides are developed with overlapping sequences on the ends and then joined together. The most common method is called polymerase cycling assembly (PCA): fragments hybridize at the overlapping regions and are extended, and larger fragments are created in each cycle. [2]

Once a sequence has been isolated, it must be inserted into a vector. The easiest way to do this is to cut the vector DNA using restriction enzymes; if the same enzymes were used to isolate the target sequence, then the same "overhang" sequences will be created on each end allowing for hybridization. Once the target gene has hybridized to the vector DNA, they can be joined using a DNA ligase. [2] An alternative strategy uses recombination between homologous sites on the target gene and the vector sequence, eliminating the need for restriction enzymes. [8]

Modes of delivery

There are three general categories of DNA construct delivery: physical, chemical, and viral. [4] Physical methods, which deliver the DNA by physically penetrating the cell, include microinjection, electroporation, and biolistics. [9] 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. [10] [11] Viral methods use a variety of viral vectors to deliver the DNA, including adenovirus, lentivirus, and herpes simplex virus [12]

Vector structure

In addition to the target gene, there are three important elements in a vector: an origin of replication, a selectable marker, and a multiple cloning site. An origin of replication is a DNA sequence that starts the process of DNA replication, allowing the vector to clone itself. A multiple cloning site contains binding sites for several restriction enzymes, making it easier to insert different DNA sequences into the vector. A selectable marker confers some trait that can be easily selected for in a host cell, so that it can be determined whether transformation was successful. The most common selectable markers are genes for antibiotic resistance, so that host cells without the construct will die off when exposed to the antibody and only host cells with the construct will remain. [2]

Types of DNA constructs

A commonly used plasmid vector, pET28a Addgene-plasmid-38252-sequence-48329-map.png
A commonly used plasmid vector, pET28a

Applications

DNA constructs can be used to produce proteins, including both naturally occurring proteins and engineered mutant proteins. These proteins can be used to make therapeutic products, such as pharmaceuticals and antibodies. DNA constructs can also change the expression levels of other genes by expressing regulatory sequences such as promoters and inhibitors. Additionally, DNA constructs can be used for research such as creating genomic libraries, sequencing cloned DNA, and studying RNA and protein expression. [5]

See also

Related Research Articles

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids often carry useful genes, such as for antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

A phagemid or phasmid is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. These vectors carry, in addition to the origin of plasmid replication, an origin of replication derived from bacteriophage. Unlike commonly used plasmids, phagemid vectors differ by having the ability to be packaged into the capsid of a bacteriophage, due to their having a genetic sequence that signals for packaging. Phagemids are used in a variety of biotechnology applications; for example, they can be used in a molecular biology technique called "phage display".

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

pBR322 Artificial plasmid

pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."

A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.

<span class="mw-page-title-main">Blue–white screen</span> DNA screening technique

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

P1 is a temperate bacteriophage that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages that integrate into the host DNA. P1 has an icosahedral head containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction. As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome. This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.

Synthetic genomics is a nascent field of synthetic biology that uses aspects of genetic modification on pre-existing life forms, or artificial gene synthesis to create new DNA or entire lifeforms.

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

pUC19 Plasmid cloning vector

pUC19 is one of a series of plasmid cloning vectors designed by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix and the abbreviation for the University of California, where early work on the plasmid series had been conducted. The pUC plasmids are all circular double stranded DNA about 2700 base pairs in length. The pUC plasmids are some of the most widely used cloning vectors. This is in part because cells that have successfully been transformed can be easily distinguished from those that have not based on color differences of colonies. pUC18 is similar to pUC19, but the MCS region is reversed.

The Gateway cloning method is a method of molecular cloning invented and commercialized by Invitrogen since the late 1990s, which makes use of the integration and excision recombination reactions that take place when bacteriophage lambda infects bacteria. This technology provides a fast and highly efficient way to transport DNA sequences into multi-vector systems for functional analysis and protein expression using Gateway att sites and two proprietary enzyme mixes called BP Clonase and LR Clonase. In vivo, these recombination reactions are facilitated by the recombination of attachment sites from the lambda/phage chromosome (attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites. The removal of the phage from the bacterial chromosome and the regeneration of attP and attB sites can both result from the attL and attR sites recombining under specific circumstances.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

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. 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.

<span class="mw-page-title-main">Vectors in gene therapy</span>

Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Cassette mutagenesis</span>

Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence to replace a fragment of target DNA. It uses complementary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity. It is different from methods that use single oligonucleotide in that a single gene cassette can contain multiple mutations. Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by DNA polymerase.

References

  1. ↑ Pinkert, Carl (2014). Transgenic animal technology: A laboratory handbook. Amsterdam: Elsevier. p. 692. ISBN   9780124095366.
  2. 1 2 3 4 5 6 7 8 9 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 2021-11-10
  3. 1 2 Hughes, Randall A.; Ellington, Andrew D. (January 2017). "Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology". Cold Spring Harbor Perspectives in Biology. 9 (1): a023812. doi:10.1101/cshperspect.a023812. ISSN   1943-0264. PMC   5204324 . PMID   28049645.
  4. 1 2 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
  5. 1 2 3 Glick, Bernard R.; Patten, Cheryl L. (2017). Molecular Biotechnology: Principles and Applications of Recombinant DNA. Washington DC: ASM Press.
  6. ↑ 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. PMID   344136.
  7. ↑ 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. PMID   2985470.
  8. ↑ Copeland, Neal G.; Jenkins, Nancy A.; Court, Donald L. (October 2001). "Recombineering: a powerful new tool for mouse functional genomics". Nature Reviews Genetics. 2 (10): 769–779. doi:10.1038/35093556. ISSN   1471-0064. PMID   11584293. S2CID   10916548.
  9. ↑ 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. PMID   15757758.
  10. ↑ 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. Bibcode:1987PNAS...84.7413F. doi: 10.1073/pnas.84.21.7413 . ISSN   0027-8424. PMC   299306 . PMID   2823261.
  11. ↑ 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. PMID   18265332. S2CID   46188175.
  12. ↑ 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. PMID   9804053.
  13. ↑ 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. Bibcode:2009PLoSO...4.8119S. doi: 10.1371/journal.pone.0008119 . ISSN   1932-6203. PMC   2780291 . PMID   19956581.
  14. ↑ Griffiths, Anthony J.F. (2015). Introduction To Genetic Analysis. New York: W.H. Freeman & Company. ISBN   978-1464188046.
  15. ↑ Godiska, R.; Wu, C. -C.; Mead, D. A. (2013-01-01), "Genomic Libraries", in Maloy, Stanley; Hughes, Kelly (eds.), 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
  16. ↑ 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. Bibcode:2019PNAS..11614222H. doi: 10.1073/pnas.1904428116 . ISSN   0027-8424. PMC   6628675 . PMID   31239340.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.