1) Electrophoresis is a laboratory technique where the blood serum (the fluid portion of the blood) is placed on special paper treated with agarose gel and exposed to an electric current to separate the serum protein components into five classifications by size and electrical charge, those being albumin
Gel electrophoresis is a group of techniques used by scientists to separate molecules based on physical characteristics such as size, shape, or isoelectric point. Gel electrophoresis is usually performed for analytical purposes, but may be used as a preparative technique to partially purify molecules prior to use of other methods such as mass spectrometry, PCR, cloning, DNA sequencing, or immuno-blotting for further characterization.
2) A transgenic organism is one whose genome has been subject to artificial modification. A transgenic organism may result when foreign DNA is inserted into the nucleus of a fertilized embryo.
A transgenic organism is one whose genome has been subject to artificial modification. A transgenic organism may result when foreign DNA is inserted into the nucleus of a fertilized embryo. Bacterial transformation is another example of a process that leads to a transgenic organism. Animal and Plant Transformation: The Application of Transgenic Organisms in Agriculture
Matthew B. Wheeler, Stephen K. Farrand, and Jack M. Widholm
A transgenic organism carries in all its cells a foreign gene that was inserted by laboratory techniques. Each transgenic organism is produced by introducing cloned genes, composed of deoxyribonucleic acid (DNA) from microbes, animals, or plants, into plant and animal cells. Transgenic technology affords methods that allow the transfer of genes between different species.
Animal Transformation
Through transgenic animal transformation, new genetic information is introduced into an animal in one generation without compromising or limiting the overall pool of genetic information. Transgenic animals are produced by inserting genes into embryos prior to birth. Each transferred gene is assimilated by the genetic material or chromosomes of the embryo and subsequently can be expressed in all tissues of the resulting animal. The objective is to produce animals which possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as "founder" stock to produce many offspring that carry a desirable gene or genes.
Transgenic animals have been produced by three methods: microinjection of cloned gene(s) into the pronucleus of a fertilized ovum, injection of embryonic stem cells into embryos, and exposure to retroviruses. The third method is not discussed in this article.
The first method is the one that is most widely and successfully used for producing transgenic mice. After microinjection, the recently fertilized single cell embryos are removed from the animal. Micromanipulators on a specially equipped microscope are used to grasp each embryo. A glass pipette drawn or pulled to a fine point immobilizes the embryo on one side, as shown in the photos to the right. On the opposite side, the foreign DNA is injected into the embryo's pronucleus--either of two nuclei (male or female) containing half the chromosomes of a fertilized ovum--with a second finely drawn injection needle. After the injection, the embryos are transferred back into the hormonally prepared or pseudopregnant recipient females or foster mothers. The recipients follow normal pregnancy and deliver full-term young. This method is presently the most efficient for generating transgenic animal lines: about 1 to 4 percent of the injected embryos result in a transgenic offspring.
The second method involves microinjection of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos (about 7 days postfertilization) into embryos to produce "hybrid" embryos of two or more distinct cell types. The ES cells are able to produce all tissues of an individual. Once isolated, ES cells may be grown in the lab for many generations to produce an unlimited number of identical cells capable of developing into fully formed adults. These cells may then be altered genetically before being used to produce embryos. When these transformed cells participate in the formation of sperm and eggs, the offspring that are produced will be transgenic. Results have shown this method to be promising for producing transgenic mice. Studies are presently under way at the University of Illinois Department of Animal Sciences to develop ES cell lines for livestock species such as swine, cattle, and sheep.
To produce transgenic mice, Matthew B. Wheeler microinjects DNA into the pronucleus of one-cell embryos.
Injection of cloned DNA into embryos. One-cell embryo is positioned for micro-injection into the pronucleus (left). The plasma membrane has been pierced, and the tip of the needle remains inside the pronucleus, while DNA is expelled from the needle, causing the pronucleus to swell visibly.
These methods, which enable the insertion of foreign genes into embryos, have provided the tools for producing new strains or breeds of animals that carry new, beneficial genetic information. These technologies do not produce new species but work within the established genetic framework of existing species to improve them. Some new strains developed include leaner, more feed-efficient, faster-growing swine containing additional copies of the growth hormone gene, and mice containing the regulatory elements of the human immunodeficiency virus (HIV) genome. The latter are used as a noninfectious animal model for the study of AIDS.
The scope of the information acquired from transgenic animal technology is pertinent to virtually all areas of modern agriculture and biomedical science--cancer research; immunology; developmental biology; gene expression and regulation; and models for human genetic diseases such as muscular dystrophy, Lou Gehring's disease, and sickle cell anemia. Potential applications for transgenic animals include manipulation of milk composition, growth, disease resistance, reproductive performance, and production of pharmaceutical proteins by livestock.
Plant Transformation
There has been much excitement in the last few years about our ability to genetically engineer plants using the new techniques of gene isolation and insertion. Paired with standard methodologies of plant tissue culture and plant regeneration, these new techniques allow us to construct transgenic plants that contain and express a single, well-defined gene from any source - microbe, animal, or other plant species. The transgenic plants, usually normal in appearance and character, differ from the parent only with respect to the function and influence of the inserted gene.
This directed genetic engineering of plants requires that genes of interest are available, that the gene be introduced into plant cells capable of regenerating into intact plants, and that the gene carries with it a selectable marker so that the transformed plant cells can be isolated from a large population of untransformed, normal cells. Finally, the transformed plant cell must retain its capacity to regenerate. Certain species such as tobacco and petunia regenerate plants quite easily, making transgenic plants readily obtainable. Although corn, soybean, and wheat--the primary agricultural crops of Illinois and the Midwest--are more recalcitrant to these manipulations, progress is being made toward routine transformation and regeneration of transgenic progeny of these species.
Several techniques can introduce genes into plant cells. Perhaps the most successful method involves the pathogenic bacterium Agrobacterium tumefaciens, which has the innate ability to transfer DNA to plant cells. In nature, this transfer results in formation of plant tumors (crown galls) at the infection site. Molecular biologists, however, have disarmed this bacterium and constructed domesticated strains that no longer cause tumors but transfer any DNA of interest to plant cells. The major disadvantage of the highly efficient Agrobacterium system is that it does not work with all plant species, most notably the cereals.
Other techniques use physical or chemical agents to transfer DNA into plant cells. Protoplasts, plant cells that have been stripped of their protective cell walls, will take up pure DNA when treated with certain membrane-active agents or with electroporation, a rapid pulse of high-voltage direct current. Once inside the cell, the DNA is integrated and the foreign gene will express. These two techniques largely depend upon the development of protoplast systems that retain the capacity to regenerate intact plants. Transgenic corn, rice, and soybean have been produced with these techniques, especially electroporation. Success rates, however, are low, and the techniques not very reproducible.
DNA can also be microinjected into target plant cells using very thin glass needles in a method similar to that used with animals. Microinjection, however, has produced only a few transgenic plants. The technique is laborious, technically difficult, and limited to the number of cells actually injected.
Biolistics, a new method, involves accelerating very small particles of tungsten or gold coated with DNA into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the particles pass through the cell, the DNA dissolves and becomes free to integrate into the plant-cell genome. This improbable technique actually works quite well and has become, along with electroporation, one of the methodologies of choice. Biolistics has the advantage of being applicable to whole cells in suspension or to intact or sliced plant tissues. For example, plant meristems or tissues capable of regeneration can be targeted directly. Unlike transformation or electroporation, the technique does not require protoplasts or even single-cell isolations. Using biolistics, transgenic corn and soybean plants have been produced that contain heritable copies of the inserted gene.
Only a few genes of agronomic importance have been inserted into plants: genes conferring resistance to certain insects and viruses and also those conferring tolerance to broad-spectrum herbicides. The latter result in increased herbicide specificity, allowing the farmer to use more effective, environmentally safe chemical agents. More recently, a gene has been introduced into tomato that delays overripening and prolongs shelf life of the fruit.
Other traits of interest include those associated with grain quality. Genes to increase the content of amino acids such as lysine, methionine, and tryptophan in seed will increase nutritional value, thereby decreasing the need for amending grains with costly feed supplements.
All traits discussed here are associated with expression of single genes. But many important agronomic traits such as yield and lodging are not well understood and are controlled by many genes. Manipulating such polygenic traits by genetic engineering will require further research and the development of techniques for isolating, reconstructing, and transferring complex blocks of genes. Extensive and promising research is being conducted about additive disease resistance and stress tolerance, important polygenic traits. Plant genetic engineering is thus moving slowly but steadily from the laboratory bench into the field.
Matthew B. Wheeler, assistant professor of animal sciences; Stephen K. Farrand, professor of plant pathology and microbiology; and Jack M. Widholm, professor of plant physiology, Department of Agronomy
TRANSGENESIS
Transgenesis
The use of recombinant DNA techniques to introduce new characters (ie. genes) into organisms (including humans) that were not present previously.
The term transgenic animal refers to an animal in which there has been a deliberate modification of the genome, in contrast to spontaneous mutation. Foreign DNA is introduced into the animal, using recombinant DNA technology, and then must be transmitted through the germ line so that every cell, including germ cells, of the animal contain the same modified genetic material.
If the germ cell line is altered, characters will be passed on to succeeding generations in normal reproduction.
If the somatic cell line alone is altered, only the organism itself will be affected, not its offspring.
Transgenesis may involve whole organisms, rather than individual cells, and there may be in vivo alteration of body function.
One use of transgenesis is gene therapy which is the alteration of the genetic make-up of of an individual organism in an attempt to correct an inborn error of metabolism, ie. cure inherited diseases. But this is generally only carried out with somatic cells and, therefore, will only affect one generation.
Do not confuse transgenesis with cloning which is the production of identical copies of molecules, cells or whole organisms. Cloning does not necessarily involve gene manipulation.
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Historical background
During the 1970s, the first chimeric mice were produced (Brinster, 1974). The cells of two different embryos of different strains were combined together at an early stage of development (eight cells) to form a single embryo that subsequently developed into a chimeric adult, exhibiting characteristics of each strain. Subsequently this was done with other animals, eg. sheep and goat ----> "geep" (1982).
Transgenesis was first described in 1981 (Gordon and Ruddle, 1981) using DNA microinjection of mice ova. Various other species such as rats, rabbits, sheep, pigs, birds, and fish soon followed.
It has been gaining application among biotechnologists since the development of transgenic "super mice" in 1982 and the development of the first mice to produce a human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987.
Two other main techniques have been developed: those of retrovirus-mediated transgenesis (Jaenisch, 1976) and embryonic stem (ES) cell-mediated gene transfer (Gossler et al., 1986).
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Uses of transgenic organisms:
in toxicology: as responsive test animals (detection of toxicants);
in mammalian developmental genetics;
to introduce human genes into other organisms (particularly human) for the study of disease processes;
in molecular biology, the analysis of the regulation of gene expression;
in the pharmaceutical industry, the production of human pharmaceuticals in farm animals ("pharming"); targeted production of pharmaceutical proteins, drug production and product efficacy testing;
in biotechnology: as producers of specific proteins;
genetically engineered hormones to increase milk yield, meat production; genetic engineering of livestock in agriculture affecting modification of animal physiology and/or anatomy; cloning procedures to reproduce specific blood lines;
to speed up the introduction of existing characters into a strain/breed for improvement and modification;
developing animals specially created for use in xenografting, ie. modify the antigenic make-up of animals so that their tissues and organs can be used in transfusions and transplants.
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Procedure for transgenesis
The inserted DNA is known as the transgene.
Conventional recombinant DNA techniques are used to construct the transgene so that the desired gene product will be expressed in the desired location. Typical transgenes contain nucleotide sequences that correspond to the gene of interest, with all the components necessary for efficient expression of the gene, including a transcription-initiation site, the 5' untranslated region, a translation-initiation codon, the coding region, a stop codon, the 3' untranslated region, a polyadenylation site and a promoter. Different promoters can be used to cause gene expression in all tissues of the body (non-specific) or only in specific tissues:
eg.
PROMOTER
GENE EXPRESSION IN
(beta)-actin promoter many tissues of the transgenic animal
simian virus 40 T antigen promoter many tissues of the transgenic animal
adipocyte P2 promoter fat cells
myosin light-chain promoter muscle
amylase promoter acinar pancreas
insulin promoter islets of Langerhans beta cells
beta-lactoglobin promoter mammary glands
In pharming expression in the mammary glands is usually desired as this leads to the appearance of the product in the milk of the animal - very convenient.
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Introduction of exogenous DNA into animal cells
The three principal methods used for the creation of transgenic animals are DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.
1. DNA microinjection
This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981) which are the most difficult of all cells to genetically manipulate. The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The DNA construct (usually about 100 to 200 copies in 2 pl of buffer) is introduced by microinjection through a fine glass needle into the male pronucleus - the nucleus provided by the sperm before fusion with the nucleus of the egg. The diameter of the egg is 70 µm and that of the glass needle is 0.75 µm; the experimenter performs the manipulations with a binocular microscope at a magnification of 200 x. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.
Microinjection is the commonest method at present and is generally more successful with laboratory animals than farm animals.
The efficiency of microinjection is quite low:
Animal species
Number of ova
injected Number of offspring Number of transgenic
offspring
rabbit 1907 218 (11.4%) 28 (1.5%)
sheep 1032 73 (7.1%) 1 (0.1%)
pig 2035 192 (9.4%) 20 (1.0%)
Figures in parentheses are percent efficiency compared to original number of ova injected.
(after Hammer et al., 1985)
2. Embryonic stem cell-mediated gene transfer
This method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.
This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.
3. Retrovirus-mediated gene transfer
To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.
For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.
There is also fusion of host cells with membranous vesicles (eg. liposomes) containing DNA.
(Plant cells can be modified using, eg. tobacco mosaic virus or the Ti plasmid of Agrobacterium tumefaciens.)
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Some examples of the use of transgenic organisms
Studying disease
Transgenic animals have been used for simulating diseases and testing new therapies, eg. cardiovascular and neurodegenerative diseases. Animal models provide an opportunity to test methods for the prevention or delay of disease in humans. Some examples:
Genetic alteration
Method of alteration
Human disease equivalent
Introduction of mutant collagen gene into wildtype mice Nuclear microinjection of inducible minigene Osteogenesis imperfecta
Inactivation of mouse gene encoding hypoxanthine-guanine phosphoribosyl transferase (HPRT) Insertion of retrovirus into HPRT locus in embryonic stem cells HPRT deficiency
Mutation at locus for X-linked muscular dystrophy Male mutagenesis followed by identification of female carriers X-linked muscular dystrophy
Introduction of activated human ras and c-myc oncogenes Nuclear microinjection of inducible minigene Induction of malignancy
Introduction of mutant (Z)allele of human alpha -1-antitrypsin gene Microinjection of DNA fragment bearing mutant allele Neonatal hepatitis
Introduction of HIV tat gene Microinjection of DNA fragment Kaposi's sarcoma
Introduction of beta-globin sickle gene Microinjection Sickle-cell anaemia
Introduction of mouse renin gene Microinjection Hypertension
Introduction of (beta)-amyloid protein precursor (APP gene)
Microinjection
Alzheimer's disease
Improving plants
Transgenic methods have now been developed for a number of important crop plants such as rice, cotton, soybean, oilseed rape and a variety of vegetable crops like tomato, potato, cabbage and lettuce. New plant varieties have been produced using bacterial or viral genes that confer tolerance to insect or disease pests and allow plants to tolerate herbicides, making the herbicide more selective in its action against weeds and allowing farmers to use less herbicide.
A new variety of cotton, for example, has been developed that uses a gene from the bacterium Bacillus thuringiensis to produce a protein that is specifically toxic to certain insect pests including bollworm, but not to animals or humans. (This protein has been used as a pesticide spray for many years.) These transgenic plants should help reduce the use of chemical pesticides in cotton production, as well as in the production of many other crops which could be engineered to contain the Bacillus thuringiensis gene. In another case, a gene from the potato leaf-roll virus has been introduced into a potato plant, giving the plant resistance to this serious potato disease.
Transgenic technologies are now being used to modify other important characteristics of plants such as the nutritional value of pasture crops or the oil quality of oilseed plants like linseed or sunflower.
Improving livestock
The main aim in using transgenic technology in animal agriculture is to improve livestock by altering their biochemistry, their hormonal balance or their important protein products. Scientists hope to produce animals that are larger and leaner, grow faster and are more efficient at using feed, more productive, or more resistant to disease. Examples of transgenic breeding programs include:
producing faster-growing and leaner pigs that use food more efficiently and resist common diseases
breeding transgenic sheep that grow better wool without needing dietary supplements of sulphur-containing amino acids.
The welfare of the animals, including any changes in metabolism that may cause health problems, is an important consideration in these programmes. Both researchers and regulatory bodies also examine closely any changes in the composition of meat or other products that will ultimately be eaten.
Advantages of transgenesis over selective breeding for animals and plants
Transgenic technology is an extension of agricultural practices that have been used for centuries: selective breeding and special feeding or fertilization programmes. It may reduce or even replace the large-scale use of pesticides and long-lasting herbicides. When fully developed, it would offer a number of advantages over traditional methods.
Compared with traditional methods, transgenic breeding is:
More specific — scientists can choose with greater accuracy the trait they want to establish. The number of additional unwanted traits can be kept to a minimum.
Faster — establishing the trait takes only one generation compared with the many generations often needed for traditional selective breeding, where much is left to chance.
More flexible — traits that would otherwise be unavailable in some animals or plants may be achievable using transgenic methods.
Less costly — much of the cost and labour involved in administering feed supplements and chemical treatments to animals and crops could be avoided.
Pharming
Many valuable pharmaceutical products can now be made using transgenic animals such as mice, rabbits, sheep, goats, pigs and cows. Often the product conveniently appears in the milk of the animal. Transgenic plants can also be used to make pharmaceuticals.
Some examples:
factor VIII blood clotting factor
factor IX blood clotting factor
fibrinogen blood clotting factor
lactoferrin as an infant formula additive
haemoglobin as a blood substitute
human protein C anticoagulant
alpha-1-antitrypsin (AAT) for treatment of AAT deficiency
tissue plasminogen activator (tPA)
cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of CF
insulin for diabetes treatment
growth hormones for treatment of deficiencies
monoclonal antibodies
vaccines (antigens)
cholesterol oxidase
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References
Brinster, R. (1974). The effect of cells transferred into mouse blastocyst on subsequent development. J. Exp. Med.:1049-1056.
Donnelly, S., McCarthy, C.R. and Singleton, R. Jr. (1994). The Brave new World of Animal Biotechnology, Special Supplement, Hastings Center Report.
Federation of European Laboratory Animal Science Associations (FELASA) September 1992, revised February 1995. Transgenic Animals - Derivation, Welfare, Use and Protection.
Gordon, J.W. and Ruddle, F.H. (1981). Integration and stable germ line transformation of genes injected into mouse pronuclei. Science 214:1244-1246
Gossler, A. et al. (1986). Transgenesis by means of blastocyst-derived embryonic stem cell line. Proc. Natl. Acad. Sci. 83:9065-9069.
Jaenisch, R. (1976). Germ line integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. 73:1260-1264.
Moore, C.J. and Mepham, T.B. (1995). Transgenesis and animal welfare. ATLA 23:380-397.
US Congress, Office of Technology Assessment (1989). New Developments in Biotechnology: Patenting Life. Special Report OTA-BA-370. 3pp. Washington DC: US Printing Office.
For Further Reading
"See How They (Don't) Grow." Successful Farming. March 1991, p. 33.
"Transgenic Animals in the Production of Therapeutic Proteins." Biotechnology International. Century Press, 1992, p. 317.
"Transgenic Pharming Advances." Bio/Technology. May 1992, p. 498.
"Whole Animals for Wholesale Protein Production." Bio/Technology. August 1992, p. 863
Transgenesis Techniques: Principles & Protocols, Vol. 180
By Alan R. Clarke
Hardcover / January 2002 / 0896036960
Germ Cell Protocols, Volume 2--Molecular Embryo Analysis, Live Imaging, Transgenesis & Cloning
By Heide Schatten
Hardcover / January 2004 / 1588292576
Animal Transgenesis & Cloning
By Louis Marie Houdebine, Gail Wagman
Paperback / April 2003 / 0470848286
Book Review
Animal Transgenesis & Cloning
By Louis Marie Houdebine
Hardcover / April 2003 / 0470848278
Book Review
Genetically Modified Organisms: Transgenesis in Plants
By Yves Tourte
Hardcover / January 2003 / 1578082609
Transgenesis: Applications of Gene Transfer
By James A. H. Murray
Hardcover / January 1994 / 0471932949
3) PCR (Polymerase Chain Reaction) is a key technique in molecular genetics that permits the analysis of any short sequence of DNA (or RNA) without having to clone it.
Polymerase chain reaction (PCR) is a molecular biology technique [1] for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA and it can be extensively modified to perform a wide array of genetic manipulations.
PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.
PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993.
As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."
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Polymerase chain reaction
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"PCR" redirects here. For other uses, see PCR (disambiguation).
Polymerase chain reaction (PCR) is a molecular biology technique [1] for enzymatically replicating DNA without using a living organism, such as E. coli or yeast. Like amplification using living organisms, the technique allows a small amount of DNA to be amplified exponentially. As PCR is an in vitro technique, it can be performed without restrictions on the form of DNA and it can be extensively modified to perform a wide array of genetic manipulations.
PCR is commonly used in medical and biological research labs for a variety of tasks, such as the detection of hereditary diseases, the identification of genetic fingerprints, the diagnosis of infectious diseases, the cloning of genes, paternity testing, and DNA computing.
PCR was invented by Kary Mullis. At the time he thought up PCR in 1983, Mullis was working in Emeryville, California for Cetus, one of the first biotechnology companies. There, he was charged with making short chains of DNA for other scientists. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway 1 one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region. Mullis has said that before his trip was over, he was already savoring the prospects of a Nobel Prize. He shared the Nobel Prize in Chemistry with Michael Smith in 1993.
As Mullis has written in the Scientific American: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat."
Contents [hide]
1 PCR in practice
1.1 Primers
1.2 Procedure
1.3 Example
1.4 PCR optimization
1.5 Difficulties with polymerase chain reaction
1.5.1 Polymerase errors
1.5.2 Size limitations
1.5.3 Non specific priming
1.6 Practical modifications to the PCR technique
1.7 Recent developments in PCR techniques
2 Uses of PCR
2.1 Genetic fingerprinting
2.2 Paternity testing
2.3 Detection of hereditary diseases
2.4 Cloning genes
2.5 Mutagenesis
2.6 Analysis of ancient DNA
2.7 Genotyping of specific mutations
2.8 Comparison of gene expression
3 History
4 Patent wars
5 References
6 External links
[edit] PCR in practice
Figure 1: A thermal cycler for PCRPCR is used to amplify specific regions of a DNA strand. This can be a single gene, just a part of a gene, or non-coding sequence. PCR typically amplifies only short DNA fragments, usually up to 10 kb. Certain methods can copy fragments up to 47 kb in size[citation needed], which is still much less than the chromosomal DNA of a eukaryotic cell - for example, a human cell contains about three billion base pairs.
PCR, as currently practiced, requires several basic components. These components are:
DNA template, which contains the region of the DNA fragment to be amplified
Two primers, which determine the beginning and end of the region to be amplified (see following section on primers)
Taq polymerase (or another durable polymerase), a DNA polymerase, which copies the region to be amplified
Deoxynucleotide triphosphates, (dNTPs) from which the DNA polymerase builds the new DNA
Buffer, which provides a suitable chemical environment for the DNA Polymerase
The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top of the reaction tubes or a layer of oil is put on the surface of the reaction mixture. These machines cost more than $2,500 USD, as of 2004.
[edit] Primers
The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.
The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer -- not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C.
Sometimes degenerate primers are used. These are actually mixtures of similar, but not identical, primers. They may be convenient if the same gene is to be amplified from different organisms, as the genes themselves are probably similar but not identical. The other use for degenerate primers is when primer design is based on protein sequence. As several different codons can code for one amino acid, it is often difficult to deduce which codon is used in a particular case. Therefore primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine. (See genetic code for further details about codons.) Use of degenerate primers can greatly reduce the specificity of the PCR amplification. This problem can be partly solved by using touchdown PCR.
The above mentioned considerations make primer design a very exacting process, upon which product yield depends:
GC-content should be between 40-60%.
Calculated Tm for both primers used in reaction should not differ >5°C and Tm of the amplification product should not differ from primers by >10°C.
Annealing temperature usually is 5°C below the calculated lower Tm. However it should be chosen empirically for individual conditions.
Inner self-complementary hairpins of >4 and of dimers >8 should be avoided.
Primer 3' terminus design is critical to PCR success since the primer extends from the 3' end. The 3' end should not be complementary over greater than 3-4 bases to any region of the other primer (or even the same primer) used in the reaction and must provide correct base matching to template.
There are computer programs to help design primers (see External links).
[edit] Procedure
The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps (Fig. 2).
The double-stranded DNA has to be heated to 94-96°C (or 98°C if extremely thermostable polymerases are used) in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: usually 1-2 minutes, but up to 5 minutes. Also certain polymerases are activated at this step (see hot-start PCR).
After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes.
Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA polymerase. Taq polymerase elongates optimally at a temperature of 72 Celsius. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, this step takes 1 minute per thousand base pairs. A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied. This differs from all other elongation steps, only in that it is longer, typically 10-15 minutes. This last step is highly recommendable if the PCR product is to be ligated into a T vector using TA-cloning.
Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94-96°C. (2) Annealing at (eg) 68°C. (3) Elongation at 72°C (P=Polymerase). (4) 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.
[edit] Example
The times and temperatures given in this example are taken from a PCR program that was successfully used on a 250 bp fragment of the C-terminus of the insulin-like growth factor (IGF)[citation needed].
Gel electrophoresis image of a standard PCR. Two sets of specific primers were used to amplify one gene from three seperate tissues. As the gel shows, Tissue #1 lacks that gene, whereas Tissue #2 and #3 possess that gene.The reaction mixture consists of
1.0 µl DNA template (100 ng/µl)
2.5 µl of primer, 1.25 µl per primer (100 ng/µl)
1.0 µl Pfu-Polymerase
1.0 µl nucleotides
5.0 µl buffer solution
89.5 µl water
A 200 µl reaction tube containing the 100 µl mixture is inserted into the thermocycler.
The PCR process consists of the following steps:
Initialization. The mixture is heated at 96°C for 5 minutes to ensure that the DNA strands as well as the primers have melted. The DNA Polymerase can be present at initialization, or it can be added after this step.
Melting, where it is heated at 96°C for 30 seconds. For each cycle, this is usually enough time for the DNA to denature.
Annealing by heating at 68°C for 30 seconds:The primers are jiggling around, caused by the Brownian motion. Short bondings are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the Tm of the double-stranded region between the template and the primer is greater than the annealing or extension temperature.
Elongation by heating 72°C for 45 seconds:This is the ideal working temperature for the polymerase. The primers, having been extended for a few bases, already have a stronger hydrogen bond to the template than the forces breaking these attractions. Primers that are on positions with no exact match, melt away from the template (because of the higher temperature) and are not extended.
The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)
Steps 2-4 are repeated 25 times, but with good primers and fresh polymerase, 15 to 20 cycles is sufficient.
Mixture is held at 7°C. This is useful if one starts the PCR in the evening just before leaving the lab, so it can run overnight. The DNA will not be damaged at 7°C after just one night.
The PCR product can be identified by its size using agarose gel electrophoresis. Agarose gel electrophoresis is a procedure that consists of injecting DNA into agarose gel and then applying an electric current to the gel. As a result, the smaller DNA strands move faster than the larger strands through the gel toward the positive current. The size of the PCR product can be determined by comparing it with a DNA ladder, which contains DNA fragments of known size, also within the gel (Fig. 3).
[edit] PCR optimization
Since PCR is very sensitive, adequate measures to avoid contamination from other DNA present in the lab environment (bacteria, viruses, lab staff's skin etc.) should be taken. Thus DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, should be performed in separate areas. For the preparation of reaction mixture, a laminar flow cabinet with UV lamp is recommended. Fresh gloves should be used for each PCR step as well as displacement pipettes with aerosol filters. The reagents for PCR should be prepared separately and used solely for this purpose. Aliquots should be stored separately from other DNA samples. A control reaction (inner control), omitting template DNA, should always be performed, to confirm the absence of contamination or primer multimer formation.
[edit] Difficulties with polymerase chain reaction
Polymerase chain reaction is not perfect, and errors and mistakes can occur. These are some common errors and problems that may occur.
[edit] Polymerase errors
Taq polymerase lacks a 3' to 5' exonuclease activity. This makes it impossible for it to check the base it has inserted and remove it if it is incorrect, a process common in higher organisms. This in turn results in a high error rate of approximately 1 in 10,000 bases, which, if an error occurs early, can alter large proportions of the final product.
Other polymerases are available for accuracy in vital uses such as amplification for sequencing. Examples of polymerases with 3'to 5' exonuclease activity include: KOD DNA polymerase, a recombinant form of Thermococcus kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis; Pfu DNA polymerase, which is extracted from Pyrococcus furiosus; and Pwo, which is extracted from Pyrococcus woesii.
[edit] Size limitations
PCR works readily with DNA of lengths two to three thousand basepairs, but above this length the polymerase tends to fall off and the typical heating cycle does not leave enough time for polymerisation to complete. It is possible to amplify larger pieces of up to 50,000 base pairs, with a slower heating cycle and special polymerases. These special polymerases are often polymerases fused to a DNA-binding protein, making them literally "stick" to the DNA longer.
[edit] Non specific priming
The non specific binding of primers is always a possibility due to sequence duplications, non-specific binding and partial primer binding, leaving the 5' end unattached. This is increased by the use of degenerate sequences or bases in the primer. Manipulation of annealing temperature and magnesium ion (which stabilise DNA and RNA interactions) concentrations can increase specificity. Non-specific priming can be prevented during the low temperatures of reaction preparation by use of "hot-start" polymerase enzymes where the active site is blocked by an antibody or chemical that only dislodges once the reaction is heated to 95˚C during the denaturation step of the first cycle.
Other methods to increase specificity include Nested PCR and Touchdown PCR.
[edit] Practical modifications to the PCR technique
Nested PCR - Nested PCR is intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. Two sets of primers are used in two successive PCR runs, the second set intended to amplify a secondary target within the first run product. This is very successful, but requires more detailed knowledge of the sequences involved.
Ligation-mediated PCR
Inverse PCR - Inverse PCR is a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of digestions and self ligation before cutting by an endonuclease, resulting in known sequences at either end of the unknown sequence.
RT-PCR - RT-PCR (Reverse Transcription PCR) is the method used to amplify, isolate or identify a known sequence from a cell or tissues RNA library. Essentially normal PCR preceded by transcription by Reverse transcriptase (to convert the RNA to cDNA) this is widely used in expression mapping, determining when and where certain genes are expressed.
Assembly PCR - Assembly PCR is the completely artificial synthesis of long gene products by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments serve to order the PCR fragments so that they selectively produce their final product.
Asymmetric PCR - Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is ideal. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.
Quantitative PCR - Q-PCR (Quantitative PCR) is used to rapidly measure the quantity of PCR product (preferably real-time), thus is an indirect method for quantitatively measuring starting amounts of DNA, cDNA or RNA. This is commonly used for the purpose of determining whether a sequence is present or not, and if it is present the number of copies in the sample. There are 3 main methods which vary in difficulty and detail.
Quantitative real-time PCR is often confusingly known as RT-PCR (Real Time PCR) and RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR can also refer to reverse transcription PCR, which even more confusingly, is often used in conjunction with Q-PCR. This method uses fluorescent dyes and probes to measure the amount of amplified product in real time.
Touchdown PCR - Touchdown PCR is a variant of PCR that reduces nonspecific primer annealing by more gradually lowering the annealing temperature between cycles. As higher temperatures give greater specificity for primer binding, primers anneal first as the temperature passes through the zone of greatest specificity.
Hot-start PCR is a technique that reduces non-specific priming that occurs during the preparation of the reaction components. The technique may be performed manually by simply heating the reaction components briefly at the melting temperature before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step.
Colony PCR - Bacterial clones (E.coli) can be screened for the correct ligation products. Selected colonies are picked with a sterile toothpick from an agarose plate and dabbed into the master mix or sterile water. Primers (and the master mix) are added - the PCR protocol has to be started with an extended time at 95^^C.
RACE-PCR - Rapid amplification of cDNA ends.
Multiplex-PCR - The use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be elicited from a single test run that otherwise would require several times the reagents and technician time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and amplicon sizes should be separated by enough difference in final base pair length to form distinct bands via gel electrophoresis.
Methylation Specific PCR - Methylation Specific PCR (MSP) is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCR reactions are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.
[edit] Recent developments in PCR techniques
A more recent method which excludes a temperature cycle, but uses enzymes, is helicase-dependent amplification.
TAIL-PCR, developed by Liu et al. in 1995, is the thermal asymmetric interlaced PCR.
Meta-PCR, developed by Andrew Wallace, allows to optimize amplification and direct sequence analysis of complex genes. Details at National Genetic Reference Laboratory, Manchester, UK
[edit] Uses of PCR
PCR can be used for a broad variety of experiments and analyses. Some examples are discussed below.
[edit] Genetic fingerprinting
Genetic fingerprinting is a forensic technique used to identify a person by comparing his or her DNA with a given sample, such as blood from a crime scene can be genetically compared to blood from a suspect. The sample may contain only a tiny amount of DNA, obtained from a source such as blood, semen, saliva, hair, or other organic material. Theoretically, just a single strand is needed. First, one breaks the DNA sample into fragments, then amplifies them using PCR. The amplified fragments are then separated using gel electrophoresis. The overall layout of the DNA fragments is called a DNA fingerprint. Since there is a very small possibility that two individuals may have the same sequences, the technique is more effective at acquitting a suspect than proving the suspect guilty. This small possibility was exploited by defense lawyers in the controversial O.J. Simpson case. A match however usually remains a very strong indicator also in the question of guilt.
[edit] Paternity testing
Figure 4: Electrophoresis of PCR-amplified DNA fragments. (1) Father. (2) Child. (3) Mother. The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new, unique fingerprint.Although these resulting 'fingerprints' are unique (except for identical twins), genetic relationships, for example, parent-child or siblings, can be determined from two or more genetic fingerprints, which can be used for paternity tests (Fig. 4). A variation of this technique can also be used to determine evolutionary relationships between organisms.
[edit] Detection of hereditary diseases
The detection of hereditary diseases in a given genome is a long and difficult process, which can be shortened significantly by using PCR. Each gene in question can easily be amplified through PCR by using the appropriate primers and then sequenced to detect mutations.
Viral diseases, too, can be detected using PCR through amplification of the viral DNA. This analysis is possible right after infection, which can be from several days to several months before actual symptoms occur. Such early diagnoses give physicians a significant lead in treatment.
[edit] Cloning genes
Cloning a gene, not to be confused with cloning a whole organism, describes the process of isolating a gene from one organism and then inserting it into another organism (now termed a genetically modified organism (GMO)). PCR is often used to amplify the gene, which can then be inserted into a vector (a vector is a piece of DNA which 'carries' the gene into the GMO) such as a plasmid (a circular DNA molecule) (Fig. 5). The DNA can then be transferred into an organism (the GMO) where the gene and its product can be studied more closely. Expressing a cloned gene (when a gene is expressed the gene product (usually protein or RNA) is produced by the GMO) can also be a way of mass-producing useful proteins, for example medicines or the enzymes in biological washing powders. The incorporation of an affinity tag on a recombinant protein will generate a fusion protein which can be more easily purified by affinity chromatography.
Figure 5: Cloning a gene using a plasmid.
(1) Chromosomal DNA of organism A. (2) PCR. (3) Multiple copies of a single gene from organism A. (4) Insertion of the gene into a plasmid. (5) Plasmid with gene from organism A. (6) Insertion of the plasmid in organism B. (7) Multiplication or expression of the gene, originally from organism A, occurring in organism B.
[edit] Mutagenesis
Mutagenesis is a way of making changes to the sequence of nucleotides in the DNA. There are situations in which one is interested in mutated (changed) copies of a given DNA strand, for example, when trying to assess the function of a gene or in in-vitro protein evolution (also known as Directed evolution). Mutations can be introduced into copied DNA sequences in two fundamentally different ways in the PCR process. Site-directed mutagenesis allows the experimenter to introduce a mutation at a specific location on the DNA strand. Usually, the desired mutation is incorporated in the primers used for the PCR program. Random mutagenesis, on the other hand, is based on the use of error-prone polymerases in the PCR process. In the case of random mutagenesis, the location and nature of the mutations cannot be controlled. One application of random mutagenesis is to analyze structure-function relationships of a protein. By randomly altering a DNA sequence, one can compare the resulting protein with the original and determine the function of each part of the protein.
[edit] Analysis of ancient DNA
Using PCR, it becomes possible to analyze DNA that is thousands of years old. PCR techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.
[edit] Genotyping of specific mutations
Through the use of allele-specific PCR, one can easily determine which allele of a mutation or polymorphism an individual has. Here, one of the two primers is common, and would anneal a short distance away from the mutation, while the other anneals right on the variation. The 3' end of the allele-specific primer is modified, to only anneal if it matches one of the alleles. If the mutation of interest is a T or C single nucleotide polymorphism (T/C SNP), one would use two reactions, one containing a primer ending in T, and the other ending in C. The common primer would be the same. Following PCR, these two sets of reactions would be run out on an agarose gel, and the band pattern will tell you if the individual is homozygous T, homozygous C, or heterozygous T/C. This methodology has several applications, such as amplifying certain haplotypes (when certain alleles at 2 or more SNPs occur together on the same chromosome Linkage Disequilibrium) or detection of recombinant chromosomes and the study of meiotic recombination.
[edit] Comparison of gene expression
Researchers have used traditional PCR as a way to estimate changes in the amount of a gene's expression. Ribonucleic acid (RNA) is the molecule into which DNA is transcribed prior to making a protein, and those strands of RNA that hold the instructions for protein sequence are known as messenger RNA (mRNA). Once RNA is isolated it can be reverse transcribed back into DNA (complementary DNA to be precise, known as cDNA), at which point traditional PCR can be applied to amplify the gene, this methodology is called RT-PCR. In most cases if there is more starting material (mRNA) of a gene then during PCR more copies of the gene will be generated. When the products of the PCR process are run on an agarose gel (see Figure 3 above) a band, corresponding to a gene, will appear larger on the gel (note that the band remains in the same location relative to the ladder, it will just appear fatter or brighter). By running samples of amplified cDNA from differently treated organisms one can get a general idea of which sample expressed more of the gene of interest. A quantative RT-PCR method has been developed, it is called Real-time PCR .
[edit] History
Polymerase chain reaction was invented by Kary Mullis. He was awarded the Nobel Prize in Chemistry in 1993 for his invention, only seven years after he and his colleagues at Cetus first reduced his proposal to practice. The idea was to develop a process by which DNA could be artificially multiplied through repeated cycles of duplication driven by an enzyme called DNA polymerase.
DNA polymerase occurs naturally in living organisms. In cells it functions to duplicate DNA when cells divide in mitosis and meiosis. Polymerase works by binding to a single DNA strand and creating the complementary strand. In the first of many original processes, the enzyme was used in vitro (in a controlled environment outside an organism). The double-stranded DNA was separated into two single strands by heating it to 94°C (201°F). At this temperature, however, the DNA polymerase used at the time were destroyed, so the enzyme had to be replenished after the heating stage of each cycle. The original procedure was very inefficient, since it required a great deal of time, large amounts of DNA polymerase, and continual attention throughout the process.
Later, this original PCR process was greatly improved by the use of DNA polymerase taken from thermophilic bacteria grown in geysers at a temperature of over 110°C (230°F). The DNA polymerase taken from these organisms is stable at high temperatures and, when used in PCR, does not break down when the mixture was heated to separate the DNA strands. Since there was no longer a need to add new DNA polymerase for each cycle, the process of copying a given DNA strand could be simplified and automated.
One of the first thermostable DNA polymerases was obtained from Thermus aquaticus and was called "Taq." Taq polymerase is widely used in current PCR practice. A disadvantage of Taq is that it sometimes makes mistakes when copying DNA, leading to mutations (errors) in the DNA sequence, since it lacks 3'→5' proofreading exonuclease activity. Polymerases such as Pwo or Pfu, obtained from Archaea, have proofreading mechanisms (mechanisms that check for errors) and can significantly reduce the number of mutations that occur in the copied DNA sequence. However these enzymes polymerise DNA at a much slower rate than Taq. Combinations of both Taq and Pfu are available nowadays that provide both high processivity (fast polymerisation) and high fidelity (accurate duplication of DNA).
PCR has been performed on DNA larger than 10 kilobases, however the average PCR is only several hundred to a few thousand bases of DNA. The problem with long PCR is that there is a balance between accuracy and processivity of the enzyme. Usually, the longer the fragment, the greater the probability of errors.
[edit] Patent wars
The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme is also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.
A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. Interestingly, it seems possible that the legal arguments will extend beyond the life of the original PCR and Taq polymerase patents, which expire in 2006.
[edit] References
^ The history of PCR: Smithsonian Institution Archives, Institutional History Division. Retrieved 24 June 2006.
Sambrook, Joseph, and David W. Russell (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. ISBN 0-87969-576-5.
Mullis, Kary (1998). Dancing Naked in the Mind Field. New York: Pantheon Books. ISBN 0-679-44255-3.
Rabinow, Paul (1996). Making PCR: A Story of Biotechnology. Chicago: University of Chicago Press. ISBN 0-226-70146-8.
[edit] External links
Wikimedia Commons has media related to:
Polymerase chain reactionPolymerase Chain Reaction (PCR) Protocol
PCR Polymerase Chain Reaction (Animation)
PCR - Polymerase Chain Reaction Articles, news, bioinformatics, and protocols for PCR.
PCR Interactive Animation
Online simulation of PCR processes against sequenced prokaryotes.
Shockwave Animation of PCR by Dolan DNA Learning Center.
The PCR Jump Station Information and links on the polymerase chain reaction
PCR Narrated flash animation
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Categories: Articles with unsourced statements | Molecular biology | Polymerase chain reaction | Amplifiers | Hoffmann-La Roche
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