Cloning Techniques

Putting Foreign DNA into Cells

The methods used to get DNA into cells are varied (e.g., transformation, transduction, transfection, and electroporation).

Learning Objectives

Describe the methods of introducing foreign DNA into cells

Key Takeaways

Key Points

  • When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation.
  • In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.
  • Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present).

Key Terms

  • transformation: The alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic.
  • microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.

The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. 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., transformation, transduction, transfection, electroporation).

When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA, are said to be competent. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. 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.

Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). 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. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

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Electroporation: Diagram of the major components of an electroporator with cuvette loaded.

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. 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.

When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. When mammalian cells (e.g., 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.

Obtaining DNA

When cloning genomic DNA, the DNA to be cloned is extracted from the organism of interest.

Learning Objectives

Explain the methods of obtaining DNA for molecular cloning experiments and the process of creating a recombinant DNA molecule

Key Takeaways

Key Points

  • The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted.
  • 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 synthesis).
  • The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process.

Key Terms

  • DNA: A biopolymer of deoxyribonucleic acids (a type of nucleic acid) that has four different chemical groups, called bases: adenine, guanine, cytosine, and thymine.
  • cloning: The production of a cloned embryo by transplanting the nucleus of a somatic cell into an ovum.
  • cloning vector: A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, into which a foreign DNA fragment can be inserted.

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 word cloning in this context refers to the fact that the method involves the replication of a single DNA molecule starting from a single living cell to generate a large population of cells containing 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.

For cloning of genomic DNA, the DNA to be cloned is extracted from the organism of interest. Virtually any tissue source can be used (even tissues from extinct animals) as long as the DNA is not extensively degraded. The DNA is then purified using simple methods to remove contaminating proteins (extraction with phenol), RNA (ribonuclease) and smaller molecules (precipitation and/or chromatography). Polymerase chain reaction (PCR) methods are often used for amplification of specific DNA or RNA (by a process known as Reverse-Transcription or RT-PCR) sequences prior to molecular cloning using primers or short DNA sequences specific for the region of interest. 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 synthesis). 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.

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The Steps of PCR: This illustrates a PCR reaction to demonstrate how amplification leads to the exponential growth of a short product flanked by the primers. 1. Denaturing at 96°C. 2. Annealing at 68°C. 3. Elongation at 72°C. The first cycle is complete. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.

Although a very large number of host organisms and molecular cloning vectors are used, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.

The cloning vector is treated with a restriction endonuclease to cleave the DNA at the site where foreign DNA will be inserted. The restriction enzyme is chosen to generate a configuration at the cleavage site that is compatible with that at the ends of the foreign DNA. Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme. 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 not located within the gene of interest to be cloned.

The creation of recombinant DNA is in many ways the simplest step of the molecular cloning process. DNA prepared from the vector and foreign source are treated with restriction enzymes to generate fragments with ends capable of being linked to those of the vector and they are simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligase) that covalently links the ends together. This joining reaction is often termed ligation. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism for amplification (a process known as transformation ). In mammalian cell culture, the analogous process of introducing DNA into cells is commonly known as transfection. Both transformation and transfection usually require preparation of the cells through a special growth regimen and chemical treatment process that will vary with the specific species and cell types that are used. 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. When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic, typically ampicillin, that would otherwise kill the cells. Cells harboring the cloning vector will survive when exposed to the antibiotic, while those that have failed to take up cloning vector will die. The former can therefore be amplified and screened for the presence of the gene of interest in the cloning vector by restriction digest analysis.

Hosts for Cloning Vectors

The majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) as the host.

Learning Objectives

Describe the features of a typical cloning vector

Key Takeaways

Key Points

  • E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment.
  • If the DNA to be cloned is exceptionally large, then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.
  • Specialized applications may call for specialized host -vector systems.

Key Terms

  • Escherichia coli: Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms).
  • plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
  • molecular cloning: a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms.

A very large number of host organisms and molecular cloning vectors are in use, but the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coli) and a plasmid cloning vector. E. coli and plasmid vectors are in common use because they are technically sophisticated, versatile, widely available, and offer rapid growth of recombinant organisms with minimal equipment. If the DNA to be cloned is exceptionally large (hundreds of thousands to millions of base pairs), then a bacterial artificial chromosome or yeast artificial chromosome vector is often chosen.

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Classification of E. coli: Domain: Bacteria, Kingdom: Eubacteria, Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae, Genus: Escherichia, Species: E. coli.

Specialized applications may call for specialized host-vector systems. For example, if the experimentalists wish to harvest a particular protein from the recombinant organism, then an expression vector is chosen that contains appropriate signals for transcription and translation in the desired host organism. 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. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning into a specialized vector.

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–(1) an origin of DNA replication is necessary for the vector (and recombinant sequences linked to it) to replicate inside the host organism, (2) one or more unique restriction endonuclease recognition sites that serves as sites where foreign DNA may be introduced, (3) a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences, and (4) an additional gene that can be used for screening which cells contain foreign DNA.

Shuttle Vectors and Expression Vectors

An expression vector is generally a plasmid that is used to introduce a specific gene into a target cell.

Learning Objectives

Explain the structure and function of shuttle and expression vectors

Key Takeaways

Key Points

  • The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
  • Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a portable translation initiation sequence.
  • Expression vectors are used for molecular biology techniques such as site-directed mutagenesis.

Key Terms

  • plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
  • expression vector: An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell.
  • transcription: The synthesis of RNA under the direction of DNA.

An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA, and in extension, proteins. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin, which is important for the treatment of diabetes.

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The pGEX-3x Plasmid: The pGEX-3x plasmid is a popular cloning vector. Please note the presence of a multiple cloning site, a promoter, a repressor, and a selectable marker.

After expression of the gene product, the purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be purified from the proteins of the host cell. Therefore, to make the purification process easy, the cloned gene should have a tag. This tag could be histidine (His) tag or any other marker peptide.

Expression vectors are used for molecular biology techniques such as site-directed mutagenesis. Cloning vectors, which are very similar to expression vectors, involve the same process of introducing a new gene into a plasmid, but the plasmid is then added into bacteria for replication purposes. In general, DNA vectors that are used in many molecular-biology gene-cloning experiments need not result in the expression of a protein.

Expression vectors must have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).

A shuttle vector is a vector that can propagate in two different host species, hence, inserted DNA can be tested or manipulated in two different cell types. The main advantage of these vectors is that they can be manipulated in E. coli and then used in a system which is more difficult or slower to use.

Shuttle vectors can be used in both eukaryotes and prokaryotes. Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli (amplification). They can also be used for in vitro experiments and modifications such as mutagenesis and PCR. One of the most common types of shuttle vectors is the yeast shuttle vector that contains components allowing for the replication and selection in both E. coli cells and yeast cells. The E. coli component of a yeast shuttle vector includes an origin of replication and a selectable marker, such as an antibiotic resistance like beta lactamase. The yeast component of a yeast shuttle vector includes an autonomously replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker.

Bacteriophage Lambda as a Cloning Vector

Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus that infects the bacterial species Escherichia coli.

Learning Objectives

Describe the life cycle of lambda phage

Key Takeaways

Key Points

  • Lambda phage consists of a virus particle including a head (also known as a capsid), tail and tail fibers.
  • Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium.
  • The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome.

Key Terms

  • transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
  • lysogeny: the process by which a bacteriophage incorporates its nucleic acids into a host bacterium
  • Lambda phage: Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. This virus is temperate and may reside within the genome of its host through lysogeny.

Enterobacteria phage λ (lambda phage, coliphage λ) is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. This virus is temperate and may reside within the genome of its host through lysogeny.

Lambda phage consists of a virus particle including a head (also known as a capsid), tail and tail fibers. The head contains the phage’s double-stranded circular DNA genome. The phage particle recognizes and binds to its host, E. coli, causing DNA in the head of the phage to be ejected through the tail into the cytoplasm of the bacterial cell. Usually, a “lytic cycle” ensues, where the lambda DNA is replicated many times and the genes for head, tail and lysis proteins are expressed. This leads to assembly of multiple new phage particles within the cell and subsequent cell lysis, releasing the cell contents, including virions that have been assembled, into the environment. However, under certain conditions, the phage DNA may integrate itself into the host cell chromosome in the lysogenic pathway. In this state, the λ DNA is called a prophage and stays resident within the host’s genome without apparent harm to the host. The host can be termed a lysogen when a prophage is present.

Lambda phage has been of major importance in the study of specialized transduction.

Specialized transduction is the process by which a restricted set of bacterial genes are transferred to another bacterium. The genes that get transferred (donor genes) depend on where the phage genome is located on the chromosome. Specialized transduction occurs when the prophage excises imprecisely from the chromosome so that bacterial genes lying adjacent to the prophage are included in the excised DNA. The excised DNA is then packaged into a new virus particle, which delivers the DNA to a new bacterium where the donor genes can be inserted into the recipient chromosome or remain in the cytoplasm, depending on the nature of the bacteriophage. When the partially encapsulated phage material infects another cell and becomes a “prophage” (is covalently bonded into the infected cell’s chromosome), the partially coded prophage DNA is called a “heterogenote. ”

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Transduction: Transduction is the process by which DNA is transferred from one bacterium to another by a virus.It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

Vectors for Genomic Cloning and Sequencing

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell.

Learning Objectives

Differentiate between expression vectors and transcription vectors

Key Takeaways

Key Points

  • The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector.
  • The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.
  • Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

Key Terms

  • vector: A carrier of a disease-causing agent.
  • plasmids: Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell.
  • chromosomes: A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements, and other nucleotide sequences.

In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the “backbone” of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) are specifically for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert.

Insertion of a vector into the target cell is usually called transformation for bacterial cells, and transfection for eukaryotic cells, although the insertion of a viral vector is often called transduction.

Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. Modern plasmids generally have many more features, notably including a “multiple cloning site” which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert.

In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours. The vectors can be extracted from the bacteria, and the multiple cloning sites can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert. These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible.

Plasmids may be conjugative / transmissible and non-conjugative. Conjugative vectors mediate DNA transfer through conjugation and therefore spread rapidly among the bacterial cells of a population, such as the F plasmid, as well as many R and some col plasmids. Non-conjugative vectors do not mediate DNA through conjugation, such as many R and col plasmids.

Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene. This allows for the translation of the transgene through a viral promoter. However, because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene. For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome.

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A Plasmid