molecular cloning


  • Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:[3]
    • DNA replication origin is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism • one or more unique restriction endonuclease recognition sites to serve as sites where foreign DNA may be introduced
    • a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences • a tag gene that can be used to screen for cells containing the foreign DNA Cleavage of a DNA sequence containing the
    BamHI restriction site.

  • Selection of organisms containing vector sequences[edit] Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency
    process; that is, only a small fraction of the cells will actually take up DNA.

  • [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.

  • Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those
    cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.

  • Libraries may be highly complex (as when cloning complete genomic DNA from an organism) or relatively simple (as when moving a previously cloned DNA fragment into a different
    plasmid), but it is almost always necessary to examine a number of different clones to be sure that the desired DNA construct is obtained.

  • cDNA cloning is usually used to obtain clones representative of the mRNA population of the cells of interest, while synthetic DNA is used to obtain any precise sequence defined
    by the designer.

  • [11] Most modern vectors contain a variety of convenient cleavage sites that are unique within the vector molecule (so that the vector can only be cleaved at a single site)
    and are located within a gene (frequently beta-galactosidase) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process.

  • Steps In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation
    of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) Selection of organisms containing recombinant DNA, (7) Screening for clones with desired DNA inserts
    and biological properties.

  • [17] In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the
    cell through a process resembling viral infection.

  • Alternatively, if replication of the DNA in different species is desired (for example, transfer of DNA from bacteria to plants), then a multiple host range vector (also termed
    shuttle vector) may be selected.

  • The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is
    chosen (e.g.

  • 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.

  • At the level of individual genes, molecular clones are used to generate probes that are used for examining how genes are expressed, and how that expression is related to other
    processes in biology, including the metabolic environment, extracellular signals, development, learning, senescence and cell death.

  • [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.

  • In practice, it is frequently more difficult to develop an organism that produces an active form of the recombinant protein in desirable quantities than it is to clone the

  • Genome organization and gene expression[edit] Molecular cloning has led directly to the elucidation of the complete DNA sequence of the genomes of a very large number of species
    and to an exploration of genetic diversity within individual species, work that has been done mostly by determining the DNA sequence of large numbers of randomly cloned fragments of the genome, and assembling the overlapping sequences.

  • Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

  • human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to
    the antibiotic Geneticin.

  • The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro
    solution, free of living cells.

  • [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.

  • 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.

  • Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant
    DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell’s DNA in the transgenic organism.

  • Such a designed sequence may be required when moving genes across genetic codes (for example, from the mitochondria to the nucleus)[14] or simply for increasing expression
    via codon optimization.

  • Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific
    species and cell types that are used.

  • Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.

  • Using a second enzyme, DNA ligase, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA.

  • Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate the genes, or make more
    subtle mutations using regional mutagenesis or site-directed mutagenesis.

  • 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.

  • This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations, antibody probes, polymerase chain reaction, restriction
    fragment analysis and/or DNA sequencing.

  • In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase, an enzyme whose activity results in formation of a blue-colored
    colony on the culture medium that is used for this work.

  • [7][8] Overview Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms.

  • [3][12] Introduction of recombinant DNA into host organism[edit] The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host

  • The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library.

  • In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.

  • Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.

  • [3][12] Applications Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome.

  • In this case, one or more specific tissues are targeted by direct treatment or by removal of the tissue, addition of the therapeutic gene or genes in the laboratory, and return
    of the treated cells to the patient.

  • Genes cloned into expression vectors for functional cloning provide a means to screen for genes on the basis of the expressed protein’s function.

  • DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA or cDNA cloning), or in the form of synthetic DNA (artificial gene

  • [4] Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process.

  • Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme so that colonies containing transformed DNA remain colorless (white).

  • [16] In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.

  • This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.

  • Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleases, enzymes
    that could cleave DNA molecules only when specific DNA sequences were encountered.

  • The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with the ends of the foreign DNA (see DNA end).

  • foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present.


Works Cited

[‘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. ^ Jump up to:a b c d e f g h 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.
6. ^ Nathans
D, Smith HO (1975). “Restriction endonucleases in the analysis and restructuring of dna molecules”. Annual Review of Biochemistry. 44: 273–93. doi:10.1146/ PMID 166604.
7. ^ Cohen SN, Chang AC, Boyer HW, Helling RB (Nov
1973). “Construction of biologically functional bacterial plasmids in vitro”. Proceedings of the National Academy of Sciences of the United States of America. 70 (11): 3240–4. Bibcode:1973PNAS…70.3240C. doi:10.1073/pnas.70.11.3240. PMC 427208. PMID
8. ^ Jackson DA, Symons RH, Berg P (Oct 1972). “Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli”.
Proceedings of the National Academy of Sciences of the United States of America. 69 (10): 2904–9. Bibcode:1972PNAS…69.2904J. doi:10.1073/pnas.69.10.2904. PMC 389671. PMID 4342968.
9. ^ “plasmid / plasmids | Learn Science at Scitable”.
Retrieved 2017-12-06.
10. ^ Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (Sep 1992). “Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector”. Proceedings
of the National Academy of Sciences of the United States of America. 89 (18): 8794–7. Bibcode:1992PNAS…89.8794S. doi:10.1073/pnas.89.18.8794. PMC 50007. PMID 1528894.
11. ^ Pingoud A, Jeltsch A (September 2001). “Structure and function of type
II restriction endonucleases”. Nucleic Acids Research. 29 (18): 3705–3727. doi:10.1093/nar/29.18.3705. PMC 55916. PMID 11557805.
12. ^ Jump up to:a b c d e f Russell DW, Sambrook J (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor,
N.Y: Cold Spring Harbor Laboratory. ISBN 978-0-87969-576-7.
13. ^ Higuchi R, Bowman B, Freiberger M, Ryder OA, Wilson AC (1984). “DNA sequences from the quagga, an extinct member of the horse family”. Nature. 312 (5991): 282–284. Bibcode:1984Natur.312..282H.
doi:10.1038/312282a0. PMID 6504142. S2CID 4313241.
14. ^ Boominathan A, Vanhoozer S, Basisty N, Powers K, Crampton AL, Wang X, et al. (November 2016). “Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant”. Nucleic
Acids Research. 44 (19): 9342–9357. doi:10.1093/nar/gkw756. PMC 5100594. PMID 27596602.
15. ^ Plotkin JB, Kudla G (January 2011). “Synonymous but not the same: the causes and consequences of codon bias”. Nature Reviews. Genetics. 12 (1): 32–42.
doi:10.1038/nrg2899. PMC 3074964. PMID 21102527.
16. ^ Lederberg J (Feb 1994). “The transformation of genetics by DNA: an anniversary celebration of Avery, MacLeod and McCarty (1944)”. Genetics. 136 (2): 423–6. doi:10.1093/genetics/136.2.423. PMC
1205797. PMID 8150273.
17. ^ Wirth R, Friesenegger A, Fiedler S (Mar 1989). “Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation”. Molecular & General Genetics. 216 (1): 175–7. doi:10.1007/BF00332248.
PMID 2659971. S2CID 25214157.
18. ^ Oldenburg J, Dolan G, Lemm G (Jan 2009). “Haemophilia care then, now and in the future”. Haemophilia. 15 (Suppl 1): 2–7. doi:10.1111/j.1365-2516.2008.01946.x. PMID 19125934. S2CID 29118026.
19. ^ The MJ (Nov
1989). “Human insulin: DNA technology’s first drug”. American Journal of Hospital Pharmacy. 46 (11 Suppl 2): S9-11. PMID 2690608.
20. ^ Lewandowski C, Barsan W (Feb 2001). “Treatment of acute ischemic stroke”. Annals of Emergency Medicine. 37 (2):
202–16. doi:10.1067/mem.2001.111573. PMID 11174240.
21. ^ Chang MH, Chen CJ, Lai MS, Hsu HM, Wu TC, Kong MS, Liang DC, Shau WY, Chen DS (Jun 1997). “Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children.
Taiwan Childhood Hepatoma Study Group”. The New England Journal of Medicine. 336 (26): 1855–9. doi:10.1056/NEJM199706263362602. PMID 9197213.
22. ^ August JT (1997). Gene Therapy. Vol. 40. Academic Press. p. 508. ISBN 978-0-08-058132-3.
23. ^
Jump up to:a b Pfeifer A, Verma IM (2001). “Gene therapy: promises and problems”. Annual Review of Genomics and Human Genetics. 2: 177–211. doi:10.1146/annurev.genom.2.1.177. PMID 11701648.
Photo credit:’]