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:
• 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 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.
 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.
 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.
 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
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.
 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 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.
 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
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) 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.
 Overview Molecular cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms.
 Introduction of recombinant DNA into host organism 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.
 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
 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).
 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.
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