![Gene cloning](https://upload.wikimedia.org/wikipedia/commons/a/a3/Gene_cloning.svg)
Molecular cloning is a set of experimental methods in
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
that are used to assemble
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
molecules and to direct their
replication within
host organisms.
The use of the word ''
cloning
Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, cl ...
'' refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living
host
A host is a person responsible for guests at an event or for providing hospitality during it.
Host may also refer to:
Places
* Host, Pennsylvania, a village in Berks County
People
*Jim Host (born 1937), American businessman
* Michel 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
In molecular cloning, a vector is any particle (e.g., plasmids, cosmids, Lambda phages) used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vec ...
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
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
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. The idea arose that different DNA sequences could be inserted into a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
and that these foreign sequences would be carried into bacteria and digested as part of the plasmid. That is, these plasmids could serve as cloning vectors to carry genes.
Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Examples of the DNA sequences that are difficult to clone are inverted repeats, origins of replication, centromeres and telomeres. There is also a lower chance of success when inserting large-sized DNA sequences. Inserts larger than 10kbp have very limited success, but bacteriophages such as
bacteriophage λ can be modified to successfully insert a sequence up to 40 kbp.
History
Prior to the 1970s, the 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 endonucleases
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
, 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
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organ ...
, fragments generated by restriction enzymes could be joined in new combinations, termed
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
. 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
DNA 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
In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...
, and the resulting
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
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
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
organism.
Molecular cloning is similar to
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR) in that it permits the replication of 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'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
'' solution, free of living cells.
''In silico'' cloning and simulations
Before actual cloning experiments are performed in the lab, most cloning experiments are planned in a computer, using specialized software. Although the detailed planning of the cloning can be done in any text editor, together with online utilities for e.g. PCR primer design, dedicated software exist for the purpose. Software for the purpose include for example Ap
(open source), DNAStride
(open source), Serial Clone
(gratis), Collagen
(open source), an
SnapGene(commercial). These programs allow to simulate
Polymerase chain reaction, PCR reactions,
restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed ''DNA fragmentation'' (this term is used for other procedures as well). Hartl and Jones describe it this way:
...
s,
ligations, etc., that is, all the steps described below.
Steps
![Steps of Molecular Cloning](https://upload.wikimedia.org/wikipedia/commons/c/c1/Steps_of_Molecular_Cloning.png)
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.
Notably, the growing capacity and fidelity of DNA synthesis platforms allows for increasingly intricate designs in molecular engineering. These projects may include very long strands of novel DNA sequence and/or test entire libraries simultaneously, as opposed to of individual sequences. These shifts introduce complexity that require design to move away from the flat nucleotide-based representation and towards a higher level of abstraction. Examples of such tools are
GenoCAD, Teselage
(free for academia) or GeneticConstructo
(free for academics).
Choice of host organism and cloning vector
![pBR322](https://upload.wikimedia.org/wikipedia/commons/5/57/PBR322.jpg)
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 coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'') 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 A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partition ...
or
yeast artificial chromosome
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 ...
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
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for ...
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 A shuttle vector is a vector (usually a plasmid) 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 ...
) 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:
* 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 serves 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
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 the ends of the foreign DNA (see
DNA end
DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...
). Typically, this is done by cleaving the vector DNA and foreign DNA with the same restriction enzyme, for example
EcoRI
''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species '' E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modific ...
. 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
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β- ...
) 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
The enzyme alkaline phosphatase (EC 3.1.3.1, alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), systematic ...
) that dephosphorylates the vector ends. Vector molecules with dephosphorylated ends are unable to replicate, and replication can only be restored if foreign DNA is integrated into the cleavage site.
Preparation of DNA to be cloned
![PCR simple](https://upload.wikimedia.org/wikipedia/commons/0/05/PCR_simple.png)
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
This page features lists of extinct species, organisms that have become extinct, either in the wild or completely disappeared from Earth.
In actual theoretical practice, a species not definitely located in the wild in the last fifty years of cur ...
),
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
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR) methods are often used for amplification of specific DNA or RNA (
RT-PCR
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase ch ...
) sequences prior to molecular cloning.
DNA for cloning experiments may also be obtained from RNA using reverse transcriptase (
complementary DNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a spe ...
or cDNA cloning), or in the form of synthetic DNA (
artificial gene synthesis
Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides '' de novo''. Unlike DNA synthesis in living cells, artificial gene synthesis do ...
). 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. Such a designed sequence may be required when moving genes across
genetic code
The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
s (for example, from the mitochrondria to the nucleus) or simply for increasing expression via
codon optimization
Codon usage bias refers to differences in the frequency of occurrence of Synonymous substitution, synonymous codons in coding DNA. A codon is a series of three nucleotides (a triplet) that encodes a specific amino acid residue in a polypeptide cha ...
.
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 ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organ ...
) that covalently links the ends together. This joining reaction is often termed
ligation
Ligation may refer to:
* Ligation (molecular biology), the covalent linking of two ends of DNA or RNA molecules
* In medicine, the making of a ligature (tie)
* Chemical ligation, the production of peptides from amino acids
* Tubal ligation, a meth ...
. 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 in 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.
transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Trans ...
,
transduction,
transfection
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to desc ...
,
electroporation
Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...
).
When microorganisms are able to take up and replicate DNA from their local environment, the process is termed
transformation
Transformation may refer to:
Science and mathematics
In biology and medicine
* Metamorphosis, the biological process of changing physical form after birth or hatching
* Malignant transformation, the process of cells becoming cancerous
* Trans ...
, 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
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to desc ...
. 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, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introdu ...
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.
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 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, a ...
is usually a gene that confers resistance to an
antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections, and antibiotic medications are widely used in the treatment and prevention of ...
that would otherwise kill the cells, typically
ampicillin
Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B strepto ...
. Cells harboring the plasmid will survive when exposed to the antibiotic, while those that have failed to take up plasmid 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
G418 (Geneticin) is an aminoglycoside antibiotic similar in structure to gentamicin B1. It is produced by '' Micromonospora rhodorangea''. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic ce ...
.
Screening for clones with desired DNA inserts and biological properties
Modern bacterial cloning vectors (e.g.
pUC19
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the abbreviation for the University of California, where early ...
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
β-Galactosidase (EC 3.2.1.23, lactase, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase), is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
β- ...
, 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 transformed DNA 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
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
,
restriction fragment analysis and/or
DNA sequencing
DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. Th ...
.
Applications
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 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. Further, cloning can be used to produce gene therapies for the treatment of serious disease indications, such as cystic fibrosis, cancer, AIDS and others. It is interesting to note that gene cloning can be a potential solution to organ scarcity. It also plays an important role in synthesis of antibiotics, vitamins and hormones.
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
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
. Genes cloned into expression vectors for
functional cloning
Functional cloning is a molecular cloning technique that relies on prior knowledge of the encoded protein’s sequence or function for gene identification. In this assay, a genomic or cDNA library is screened to identify the genetic sequence of a p ...
provide a means to screen for genes on the basis of the expressed protein's function.
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 (FVIII) is an essential blood-clotting protein, also known as anti-hemophilic factor (AHF). In humans, factor VIII is encoded by the ''F8'' gene. Defects in this gene result in hemophilia A, a recessive X-linked coagulation disorder. ...
, a blood-clotting factor deficient in some forms of
hemophilia
Haemophilia, or hemophilia (), is a mostly inherited genetic disorder that impairs the body's ability to make blood clots, a process needed to stop bleeding. This results in people bleeding for a longer time after an injury, easy bruising, ...
,
and recombinant
insulin
Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism o ...
, used to treat some forms of
diabetes
Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ap ...
), (2) proteins that can be administered to assist in a life-threatening emergency (e.g.
tissue plasminogen activator
Tissue plasminogen activator (abbreviated tPA or PLAT) 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 ...
, 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 is a vaccine that prevents hepatitis B. The first dose is recommended within 24 hours of birth with either two or three more doses given after that. This includes those with poor immune function such as from HIV/AIDS and th ...
), 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
A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
(GMOs). Although most GMOs are generated for purposes of basic biological research (see for example,
transgenic mouse
A genetically modified mouse or genetically engineered mouse model (GEMM) is a mouse (''Mus musculus'') that has had its genome altered through the use of genetic engineering techniques. Genetically modified mice are commonly used for research or ...
), a number of GMOs have been developed for commercial use, ranging from animals and plants that produce pharmaceuticals or other compounds (
pharming
Pharming is a cyberattack intended to redirect a website's traffic to another, fake site by installing a malicious program on the computer. Pharming can be conducted either by changing the hosts file on a victim's computer or by exploitation of a ...
),
herbicide-resistant crop plants, and fluorescent tropical fish (
GloFish
The GloFish is a patented and trademarked brand of genetically engineered fluorescent fish. They have been created from several different species of fish: zebrafish (''Danio rerio'') were the first GloFish available in pet stores, and recently te ...
) 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.
References
Further reading
*
External links
*
{{DEFAULTSORT:Molecular Cloning
Genetics techniques
Molecular genetics
Molecular biology techniques