Molecular cloning
Encyclopedia
Molecular cloning refers to 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 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
. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
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. Subsequently, these fragments are then combined with vector DNA
to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically-modified microorganisms (GMO
). 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. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.
, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme, DNA ligase
, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA
. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972.
is fundamentally the same in all living 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.
Molecular cloning is similar to polymerase chain reaction
(PCR) in that it permits the replication of a specific DNA sequence. 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.
) 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.
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.
). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI
. 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 is 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. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase
) that modifies the vector ends in such a way that it cannot replicate within cells unless it contains foreign DNA.
), 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 (RT-PCR
) sequences prior to molecular cloning.
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.
The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector.
) 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.
DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.
, 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.
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 confers resistance to the antibiotic Geneticin.
and later derivatives including the pGEM vectors) use the blue-white screening system
to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). 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. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing recombinant plasmids remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.
The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. 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. 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
.
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. 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
.
Many useful proteins are currently available as recombinant products. These include--(1) medically-useful proteins whose administration can correct a defective or poorly-expressed gene (e.g. recombinant factor VIII
, a blood-clotting factor deficient in some forms of hemophilia, and recombinant insulin
, used to treat some forms of diabetes), (2) proteins that can be administered to assist in a life threatening emergency (e.g. tissue plasminogen activator
, used to treat strokes), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g. hepatitis B vaccine
), and (4) recombinant proteins as standard material for diagnostic laboratory tests.
), herbicide-resistant crop plants, and fluorescent tropical fish (GloFish
) for home entertainment.
Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
that are used to assemble recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
molecules and to direct their replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
within host organisms
Host (biology)
In biology, a host is an organism that harbors a parasite, or a mutual or commensal symbiont, typically providing nourishment and shelter. In botany, a host plant is one that supplies food resources and substrate for certain insects or other fauna...
. The use of the word cloning
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
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
Host (biology)
In biology, a host is an organism that harbors a parasite, or a mutual or commensal symbiont, typically providing nourishment and shelter. In botany, a host plant is one that supplies food resources and substrate for certain insects or other fauna...
for replication of the recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
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. Subsequently, these fragments are then combined with vector DNA
Vector (molecular biology)
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, viruses, cosmids, and artificial chromosomes...
to generate recombinant DNA molecules. The recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA. Because they contain foreign DNA fragments, these are transgenic or genetically-modified microorganisms (GMO
Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
). 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. This single cell can then be expanded exponentially to generate a large amount of bacteria, each of which contain copies of the original recombinant molecule. Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones". Strictly speaking, recombinant DNA refers to DNA molecules, while molecular cloning refers to the experimental methods used to assemble them.
History of molecular cloning
Prior to the 1970s, our understanding of genetics and molecular biology was severely hampered by an inability to isolate and study individual genes from complex organisms. This changed dramatically with the advent of molecular cloning methods. Microbiologists, seeking to understand the molecular mechanisms through which bacteria restricted the growth of bacteriophage, isolated restriction endonucleasesRestriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
, enzymes that could cleave DNA molecules only when specific DNA sequences were encountered. They showed that restriction enzymes cleaved chromosome-length DNA molecules at specific locations, and that specific sections of the larger molecule could be purified by size fractionation. Using a second enzyme, DNA ligase
Ligase
In biochemistry, ligase is an enzyme that can catalyse the joining of two large molecules by forming a new chemical bond, usually with accompanying hydrolysis of a small chemical group dependent to one of the larger molecules...
, fragments generated by restriction enzymes could be joined in new combinations, termed recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
. By recombining DNA segments of interest with vector DNA, such as bacteriophage or plasmids, which naturally replicate inside bacteria, large quantities of purified recombinant DNA molecules could be produced in bacterial cultures. The first recombinant DNA molecules were generated and studied in 1972.
Overview
Molecular cloning takes advantage of the fact that the chemical structure of DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
is fundamentally the same in all living organisms. Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
, and the resulting recombinant DNA
Recombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
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.
Molecular cloning is similar to polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) in that it permits the replication of a specific DNA sequence. 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
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
solution, free of living cells.
Steps in molecular cloning
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.Choice of host organism and cloning vector
Although a very large number of host organisms and molecular cloning vectors are in use, the great majority of molecular cloning experiments begin with a laboratory strain of the bacterium E. coli (Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
) 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
Bacterial artificial chromosome
A bacterial artificial chromosome 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...
or yeast artificial chromosome
Yeast artificial chromosome
A yeast artificial chromosome is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes...
vector is often chosen.
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
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. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
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
Shuttle vector
A shuttle vector is a vector constructed so that it can propagate in two different host species . Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli then used in a...
) may be selected. In practice, however, specialized molecular cloning experiments usually begin with cloning into a bacterial plasmid, followed by subcloning
Subcloning
In molecular biology, subcloning is a technique used to move a particular gene of interest from a parent vector to a destination vector in order to further study its functionality....
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.
Preparation of vector DNA
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 (see DNA endDNA end
DNA end or sticky end refers to the properties of the end of a molecule of DNA or a recombinant DNA molecule. The concept is important in molecular biology, especially in cloning or when subcloning inserts DNA into vector DNA. All the terms can also be used in reference to RNA. The sticky ends or...
). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example EcoRI
EcoRI
EcoRI is an endonuclease enzyme isolated from strains of E. coli, and is part of the restriction modification system.In molecular biology it is used as a restriction enzyme. It creates sticky ends with 5' end overhangs...
. 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 is located within a gene (frequently beta-galactosidase
Blue white screen
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
) whose inactivation can be used to distinguish recombinant from non-recombinant organisms at a later step in the process. To improve the ratio of recombinant to non-recombinant organisms, the cleaved vector may be treated with an enzyme (alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
) that modifies the vector ends in such a way that it cannot replicate within cells unless it contains foreign DNA.
Preparation of DNA to be cloned
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 animalsQuagga
The quagga is an extinct subspecies of the plains zebra, which was once found in great numbers in South Africa's Cape Province and the southern part of the Orange Free State. It was distinguished from other zebras by having the usual vivid marks on the front part of the body only...
), 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
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(PCR) methods are often used for amplification of specific DNA or RNA (RT-PCR
Reverse transcription polymerase chain reaction
Reverse transcription polymerase chain reaction is a variant of polymerase chain reaction , a laboratory technique commonly used in molecular biology to generate many copies of a DNA sequence, a process termed "amplification"...
) sequences prior to molecular cloning.
DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (complementary DNA
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
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.
The purified DNA is then treated with a restriction enzyme to generate fragments with ends capable of being linked to those of the vector. If necessary, short double-stranded segments of DNA (linkers) containing desired restriction sites may be added to create end structures that are compatible with the vector.
Creation of recombinant DNA with DNA ligase
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 simply mixed together at appropriate concentrations and exposed to an enzyme (DNA ligaseDNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
) that covalently links the ends together. This joining reaction is often termed ligation
Ligation
Ligation may refer to:* In molecular biology, the covalent linking of two ends of DNA molecules using DNA ligase* In medicine, the making of a ligature * Chemical ligation, the production of peptides from amino acids...
. The resulting DNA mixture containing randomly joined ends is then ready for introduction into the host organism.
DNA ligase only recognizes and acts on the ends of linear DNA molecules, usually resulting a complex mixture of DNA molecules with randomly joined ends. The desired products (vector DNA covalently linked to foreign DNA) will be present, but other sequences (e.g. foreign DNA linked to itself, vector DNA linked to itself and higher-order combinations of vector and foreign DNA) are also usually present. This complex mixture is sorted out in subsequent steps of the cloning process, after the DNA mixture is introduced into cells.
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 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. transformationTransformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
, transduction
Transduction (genetics)
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. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
, transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
, electroporation
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
).
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation
Transformation (genetics)
In molecular biology transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material from its surroundings and taken up through the cell membrane. Transformation occurs naturally in some species of bacteria, but it can...
, and cells that are in a physiological state such that they can take up DNA are said to be competent
Competence (biology)
In microbiology, genetics, cell biology and molecular biology, competence is the ability of a cell to take up extracellular DNA from its environment...
. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
. 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
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction
Transduction (genetics)
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. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
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.
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. 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
Selectable marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
is usually a gene that confers resistance to an antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
that would otherwise kill the cells, typically ampicillin
Ampicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
. 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 confers resistance to the antibiotic Geneticin.
Screening for clones with desired DNA inserts and biological properties
Modern bacterial cloning vectors (e.g. pUC19PUC19
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs...
and later derivatives including the pGEM vectors) use the blue-white screening system
Blue white screen
The blue-white screen is a screening technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
to distinguish colonies (clones) of transgenic cells from those that contain the parental vector (i.e. vector DNA with no recombinant sequence inserted). In these vectors, foreign DNA is inserted into a sequence that encodes an essential part of beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
, an enzyme whose activity results in formation of a blue-colored colony on the culture medium that is used for this work. Insertion of the foreign DNA into the beta-galactosidase coding sequence disables the function of the enzyme, so that colonies containing recombinant plasmids remain colorless (white). Therefore, experimentalists are easily able to identify and conduct further studies on transgenic bacterial clones, while ignoring those that do not contain recombinant DNA.
The total population of individual clones obtained in a molecular cloning experiment is often termed a DNA library. 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. This may be accomplished through a very wide range of experimental methods, including the use of nucleic acid hybridizations
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
, antibody probes
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
, polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
, restriction fragment analysis
Restriction fragment length polymorphism
In molecular biology, restriction fragment length polymorphism, or RFLP , is a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related...
and/or DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
.
Applications of molecular cloning
Molecular cloning provides scientists with an essentially unlimited quantity of any individual DNA segments derived from any genome. This material can be used for a wide range of purposes, including those in both basic and applied biological science. A few of the more important applications are summarized here.Genome organization and gene expression
Molecular cloning has led directly to the elucidation of the complete of sexual 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.At the level of individual genes, molecular clones are used to generate probes
Molecular probe
A molecular probe is a group of atoms or molecules attached to other molecules or cellular structures and used in studying the properties of these molecules and structures. Radioactive DNA or RNA sequences are used in molecular genetics to detect the presence of a complementary sequence by...
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. Cloned genes can also provide tools to examine the biological function and importance of individual genes, by allowing investigators to inactivate
Knockout mouse
A knockout mouse is a genetically engineered mouse in which researchers have inactivated, or "knocked out," an existing gene by replacing it or disrupting it with an artificial piece of DNA...
the genes, or make more subtle mutations using regional mutagenesis or site-directed mutagenesis
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
.
Production of recombinant proteins
Obtaining the molecular clone of a gene can lead to the development of organisms that produce the protein product of the cloned genes, termed a recombinant protein. 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 gene. This is because the molecular signals for gene expression are complex and variable, and because protein folding, stability and transport can be very challenging.Many useful proteins are currently available as recombinant products. These include--(1) medically-useful proteins whose administration can correct a defective or poorly-expressed gene (e.g. recombinant factor VIII
Factor VIII
Factor VIII is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...
, a blood-clotting factor deficient in some forms of hemophilia, and recombinant insulin
Humulin
Humulin is the brand name for a group of biosynthetic human insulin products, originally developed by Genentech in 1978 and later acquired by Eli Lilly and Company, the company who arguably facilitated...
, used to treat some forms of diabetes), (2) proteins that can be administered to assist in a life threatening emergency (e.g. tissue plasminogen activator
Tissue plasminogen activator
Tissue plasminogen activator is a protein involved in the breakdown of blood clots. It is a serine protease found on endothelial cells, the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown...
, used to treat strokes), (3) recombinant subunit vaccines, in which a purified protein can be used to immunize patients against infectious diseases, without exposing them to the infectious agent itself (e.g. hepatitis B vaccine
Hepatitis B vaccine
Hepatitis B vaccine is a vaccine developed for the prevention of hepatitis B virus infection. The vaccine contains one of the viral envelope proteins, hepatitis B surface antigen . It is produced by yeast cells, into which the genetic code for HBsAg has been inserted...
), and (4) recombinant proteins as standard material for diagnostic laboratory tests.
Transgenic organisms
Once characterized and manipulated to provide signals for appropriate expression, cloned genes may be inserted into organisms, generating transgenic organisms, also termed genetically-modified organisms (GMOs). Although most GMOs are generated for purposes of basic biological research (see for example, transgenic mouse), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (pharmingPharming (genetics)
Pharming is a portmanteau of farming and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes. As a consequence, the host animals or plants then make the...
), herbicide-resistant crop plants, and fluorescent tropical fish (GloFish
GloFish
The GloFish is a patented and trademarked brand of genetically modified fluorescent zebrafish with bright red, green, orange-yellow, blue, and purple fluorescent colors...
) for home entertainment.
Gene therapy
Gene therapy involves supplying a functional gene to cells lacking that function, with the aim of correcting a genetic disorder or acquired disease. Gene therapy can be broadly divided into two categories. The first is alteration of germ cells, that is, sperm or eggs, which results in a permanent genetic change for the whole organism and subsequent generations. This “germ line gene therapy” is considered by many to be unethical in human beings. The second type of gene therapy, “somatic cell gene therapy”, is analogous to an organ transplant. 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. Clinical trials of somatic cell gene therapy began in the late 1990s, mostly for the treatment of cancers and blood, liver, and lung disorders.Despite a great deal of publicity and promises, the history of human gene therapy has been characterized by relatively limited success. The effect of introducing a gene into cells often promotes only partial and/or transient relief from the symptoms of the disease being treated. Some gene therapy trial patients have suffered adverse consequences of the treatment itself, including deaths. In some cases, the adverse effects result from disruption of essential genes within the patient's genome by insertional inactivation. In others, viral vectors used for gene therapy have been contaminated with infectious virus. Nevertheless, gene therapy is still held to be a promising future area of medicine, and is an area where there is a significant level of research and development activity.