The latest study, DNA molecules appear to have telepathy. Scientists have discovered the double helix structure of DNA molecules to identify themselves with the "matching" elements, even some distance away, on the surface and no other outside help, the match of the two elements together to the end. In accordance with the previous understanding of DNA, scientists study the double helix structure of DNA molecules are arranged in accordance with the laws of their own. Helix structure of DNA molecule is composed of many ODN from the polymerization of long-chain, as the composition of the DNA base pairs of only four: adenine (A), guanine (G), cytosine (C) And thymine (T), therefore, there are four types of DNA, we usually A, T, C, G Four alphabet tag them, and use of chemical methods to match their children - A equipped with T, C equipped with G . In fact, the well-known "to exchange the role of the base together" is not the DNA double helix molecule in close connection with the bodies of the root causes. The DNA double helix structure was so stable, because the outside of deoxyribose phosphate and arranged in alternating the basic framework, the inside of the base to form hydrogen bonds through the base right. Scientists study the mixture through the fluorescence of this double-stranded DNA molecule structure. These DNA molecules were placed on some of the salt water, salt water test does not contain any proteins, DNA molecules will enable non-binding, as well as any material that may affect the trial. Strangely enough, together with the same number of base pairs of DNA molecules is the other remaining twice the number of DNA molecules. Even though they look like a very strange, like a psychological sense, but in fact only a DNA molecule in the exercise under the laws of physics, not a supernatural phenomenon. Take charge of deoxyribose and phosphate arranged in turn composed of DNA molecules will be mutually exclusive, however, because of the DNA double helix structure of the special, making the repulsive force between them to reach the minimum. In order to understand more vividly the researchers said, let us try the double helix structure of DNA molecules into a corkscrew imagination to form a long chain of DNA molecules in the base of support outside the framework and the role of hydrogen bonds in the middle of, So that the screw cone to the direction of a distorted, twisted into a spiral, then the process will be part of the same degree of bending and other elements of the sunken part of the coordinated combination. Scientists point out that this "psychological sense" would help the DNA molecules in the chaos of their pre-arranged neatly, which can effectively avoid errors occur when the combination of DNA, it effectively avoiding cancer, aging and other diseases. However, due to the same DNA sequence in fact disrupt the combination of sexual reproduction is a meaningful, because of the need to ensure that future generations have the genetic diversity.
Thursday, December 31, 2009
Tuesday, December 22, 2009
RNA extraction from tissues
Procedures for RNA extraction from tissues :
1. pre-weigh an empty cryovial
2. take the tissue specimen out from the cryovial and place it on a 100mm petri dish
3. use a scapel and sterican (like fork and knife) to cut the tissue into desired size
4. transfer the tissue into the pre-weigh cryovial and weigh again (this will give the exact weigh of the tissue being process)
5. transfer the surplus tissue into the original cryovial and keep in the nitrogen tank
6. add 650µl of RLT buffer into the cryovial
7. pound the tissue using a pipette tip until the solution turn orange in colour.
8. transfer the solution into QIA shredder column and centrifuge at 14 000rpm for 4minutes
9. transfer the flow through into a new eppendorf tube and add in 550µl of 70% ethanol
10. centrifuge the eppendorf tube at 14 000rpm for 3 minutes
11. transfer the solution into Rneasy column and centrifuge at 14 000rpm for 15 seconds
12. discard the flow through
13. add 700µl RW1 buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
14. discard the flow through
15. add 500µl RPE buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
16. discard the flow through
17. centrifuge the Rneasy column at 14 000rpm for 1 minute
18. transfer the Rneasy column into a new sterilised round bottom tube
19. add 30µl Rnase-free water and centrifuge at 14 000rpm for 1 minute
20. transfer the flow into the column and centrifuge at 14 000rpm for 1 minute
21. the flow through is the RNA extracted
Thing to note
- after taking the cryovial of tissue out from the nitrogen tank, keep it in liquid nitrogen.
1. pre-weigh an empty cryovial
2. take the tissue specimen out from the cryovial and place it on a 100mm petri dish
3. use a scapel and sterican (like fork and knife) to cut the tissue into desired size
4. transfer the tissue into the pre-weigh cryovial and weigh again (this will give the exact weigh of the tissue being process)
5. transfer the surplus tissue into the original cryovial and keep in the nitrogen tank
6. add 650µl of RLT buffer into the cryovial
7. pound the tissue using a pipette tip until the solution turn orange in colour.
8. transfer the solution into QIA shredder column and centrifuge at 14 000rpm for 4minutes
9. transfer the flow through into a new eppendorf tube and add in 550µl of 70% ethanol
10. centrifuge the eppendorf tube at 14 000rpm for 3 minutes
11. transfer the solution into Rneasy column and centrifuge at 14 000rpm for 15 seconds
12. discard the flow through
13. add 700µl RW1 buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
14. discard the flow through
15. add 500µl RPE buffer into the Rneasy column and centrifuge at 14 000rpm for 15 seconds
16. discard the flow through
17. centrifuge the Rneasy column at 14 000rpm for 1 minute
18. transfer the Rneasy column into a new sterilised round bottom tube
19. add 30µl Rnase-free water and centrifuge at 14 000rpm for 1 minute
20. transfer the flow into the column and centrifuge at 14 000rpm for 1 minute
21. the flow through is the RNA extracted
Thing to note
- after taking the cryovial of tissue out from the nitrogen tank, keep it in liquid nitrogen.
Sunday, October 25, 2009
The structure of dna
DNA is composed of many DNA residue, according to a certain order each other with 3 ', 5'-phosphate ester linked to the composition of long chain. DNA contains a majority of two such long-chain, and some for single-stranded DNA, such as E. coli phage φX174, G4, M13 and so on. Some DNA for the ring, and some for the linear DNA. Contains adenine, guanine, thymine and cytosine base 4. In some types of DNA, 5 - cytosine methylation may to a certain extent replaced by cytosine, wheat germ DNA of the 5 - cytosine-rich in particular, up to 6% of the mole. In some phage, 5 - hydroxymethyl cytosine replaced cytosine. In the late 40, Gabriel Richard (E. Chargaff) found that different species of the base composition of DNA, but the number of adenine equal to the number of its thymine (A = T), guanine cytosine number equal to the number (G = C), and therefore the number of purine and pyrimidine equal to the number of and. Several described by the general level of the structure of DNA.
A structure of the primary structure of DNA that is its base sequence. Gene is a fragment of DNA, genetic information stored in its base sequence. In 1975 the United States Gilbert (W. Gilbert) and the United Kingdom's Sanger (F. Sanger) respectively, created the structure of the DNA level, the rapid determination of their total end of 1980 Nobel Prize in Chemistry. Since then, the method also has been improved, many of the primary structure of DNA has been established. If people ring of mitochondrial DNA contains 16,569 base pairs, λ phage DNA contains 48,502 base pairs, rice chloroplast genome contains 134,525 base pairs, tobacco chloroplast genome contains 155,844 base pairs, and so on. Now the United States has plans to 10-15 years in all human DNA molecule of about 3,000,000,000 for the nucleotide sequence out.
Secondary structure in 1953, Watson (Watson) and Crick (Crick) put forward the basic structure of DNA fiber is a double helix structure, then this model has been recognized scientists, and to explain to copy, transcription, and other important life processes. After an in-depth study and found that humidity and as a result of base sequences in different conditions, DNA double helix can have a variety of types, mainly divided into A, B and Z three categories.
It is generally believed, B cells closest to the structure of DNA conformation, it is very similar to the double helix model. A-DNA and RNA molecules in the two-time transcription, as well as the district Screw the formation of the DNA-RNA hybridization close to the molecular conformation. Z-DNA nucleotide dimer to the left to the wound as a unit, which was the main chain saw (Z)-shaped, and named. This configuration for multi-purine nucleotide chains of alternating pyrimidine area. In 1989, U.S. scientists used scanning tunneling electron microscope to directly observe the double helix DNA double helix DNA ︰ 1952, Austria-American biochemist Chargaff (E.chargaff, 1905 -) was determined by DNA base pairs in the 4 Content and found that methotrexate gland and fat equal to the number of thymine, cytosine and methotrexate fat birds of the same number. This Watson, Crick immediately think of base 4 between 22 corresponds to the relationship between the formation of the gland fat and thymine pair methotrexate, methotrexate fat birds and the concept of cytosine pair.
High structure
Triplex DNA (T-DNA)
In the early 1950s, Wilkins According to the X-ray diffraction pictures have been envisaged in the DNA chain may be 3, Crick later watson and others have to build some T-DNA model. Has found that the three DNA chain can be divided into two categories, namely, three helix structure of the Chinese Academy of Sciences and in 1990 by Bai Chunli, such as scanning tunneling electron microscope techniques to observe the structure of the three Fabian "
Three in the spiral structure of DNA double helix structure formed on the basis of the three chains District 3 chain are homologous or homologous pyrimidine purine that the entire base of the pyrimidine purine or, in accordance with section 3 of the chain of sources , The DNA chain can be divided into three elements and between elements within the two groups, according to the 3 components of the chain as well as the relative positions can be divided into Pu-Pu-Py and Py-Pu-Py-two (Pu-generation
Table purine chain, Py pyrimidine on behalf of the chain) is one of the most common Py-Pu-Py type, it's 3 in the chain there are 2 for a normal double helix, 3 pyrimidine chain located in the double helix of the big ditch, Purine with the chain in the same direction and with the double helix structure of the rotation together, the three chains in the base pairs of DNA double helix with the same, that is, their base is still AT, GC matching However, No. 3 chain C must be protonated , And G with only 2 to form hydrogen bonds (normally three hydrogen bonds).
Triplex DNA research will help shed further light on the structure of chromosomes and genes in eukaryotic transcription, replication, and re-regulation and control mechanism. In addition three DNA chain have a certain value, such as the availability of single-stranded DNA fragments will be cutting agent (such as the endonuclease) to carry DNA to a specific site, so as to achieve selective chromosome DNA hit off the end, because the cells Transcription factors such as regulation and protein only after the combination of DNA double helix to open its specific gene transcription, transcription factors can not helical and the three combined, it can make use of oligo DNA fragment closure of the transcription factor binding sites in order to reach the turn off harmful genes or Virus genes.
Quadruplex DNA
Klug and Sundpuist in a simulated 1 kind of spike protozoan telomere DNA of the caterpillar, paragraph 1 of the synthetic DNA sequences and found that under certain conditions, the simulation of G-rich single-stranded DNA to form a quadruplex DNA structure. This chromosome that the end of the telomere single strand between the formation of a four-chain. Kang and others were confirmed by experiments in crystal and in solution, the rich G DNA can form a quadruplex DNA structure.
Quadruplex DNA is the basic structure of the G-quadruplex, that is, in the four conjoined at the center there are 4 by a negative charge with the carboxylic oxygen atoms surrounded the "pocket" through the G-quadruplex accumulation can be Elements to form molecules or between right-handed spiral, with the double helix structure of DNA comparison, G-quadruplex spiral 2 significant characteristics: 1, the stability of its decision in the pocket by the combination of cation type, known ion k The combination of quadruple-helix so that the most stable; 2, and its thermodynamic stability of the very nature and dynamics.
At present some of the biological DNA sequence analysis that the G-rich DNA sequence found in some of the functions and evolution are very conservative region of the genome, many studies have shown that guanine-rich DNA chain formed by G-DNA may be As the mutual recognition between the molecular components of one of the organisms in the cell plays a special role
A structure of the primary structure of DNA that is its base sequence. Gene is a fragment of DNA, genetic information stored in its base sequence. In 1975 the United States Gilbert (W. Gilbert) and the United Kingdom's Sanger (F. Sanger) respectively, created the structure of the DNA level, the rapid determination of their total end of 1980 Nobel Prize in Chemistry. Since then, the method also has been improved, many of the primary structure of DNA has been established. If people ring of mitochondrial DNA contains 16,569 base pairs, λ phage DNA contains 48,502 base pairs, rice chloroplast genome contains 134,525 base pairs, tobacco chloroplast genome contains 155,844 base pairs, and so on. Now the United States has plans to 10-15 years in all human DNA molecule of about 3,000,000,000 for the nucleotide sequence out.
Secondary structure in 1953, Watson (Watson) and Crick (Crick) put forward the basic structure of DNA fiber is a double helix structure, then this model has been recognized scientists, and to explain to copy, transcription, and other important life processes. After an in-depth study and found that humidity and as a result of base sequences in different conditions, DNA double helix can have a variety of types, mainly divided into A, B and Z three categories.
It is generally believed, B cells closest to the structure of DNA conformation, it is very similar to the double helix model. A-DNA and RNA molecules in the two-time transcription, as well as the district Screw the formation of the DNA-RNA hybridization close to the molecular conformation. Z-DNA nucleotide dimer to the left to the wound as a unit, which was the main chain saw (Z)-shaped, and named. This configuration for multi-purine nucleotide chains of alternating pyrimidine area. In 1989, U.S. scientists used scanning tunneling electron microscope to directly observe the double helix DNA double helix DNA ︰ 1952, Austria-American biochemist Chargaff (E.chargaff, 1905 -) was determined by DNA base pairs in the 4 Content and found that methotrexate gland and fat equal to the number of thymine, cytosine and methotrexate fat birds of the same number. This Watson, Crick immediately think of base 4 between 22 corresponds to the relationship between the formation of the gland fat and thymine pair methotrexate, methotrexate fat birds and the concept of cytosine pair.
High structure
Triplex DNA (T-DNA)
In the early 1950s, Wilkins According to the X-ray diffraction pictures have been envisaged in the DNA chain may be 3, Crick later watson and others have to build some T-DNA model. Has found that the three DNA chain can be divided into two categories, namely, three helix structure of the Chinese Academy of Sciences and in 1990 by Bai Chunli, such as scanning tunneling electron microscope techniques to observe the structure of the three Fabian "
Three in the spiral structure of DNA double helix structure formed on the basis of the three chains District 3 chain are homologous or homologous pyrimidine purine that the entire base of the pyrimidine purine or, in accordance with section 3 of the chain of sources , The DNA chain can be divided into three elements and between elements within the two groups, according to the 3 components of the chain as well as the relative positions can be divided into Pu-Pu-Py and Py-Pu-Py-two (Pu-generation
Table purine chain, Py pyrimidine on behalf of the chain) is one of the most common Py-Pu-Py type, it's 3 in the chain there are 2 for a normal double helix, 3 pyrimidine chain located in the double helix of the big ditch, Purine with the chain in the same direction and with the double helix structure of the rotation together, the three chains in the base pairs of DNA double helix with the same, that is, their base is still AT, GC matching However, No. 3 chain C must be protonated , And G with only 2 to form hydrogen bonds (normally three hydrogen bonds).
Triplex DNA research will help shed further light on the structure of chromosomes and genes in eukaryotic transcription, replication, and re-regulation and control mechanism. In addition three DNA chain have a certain value, such as the availability of single-stranded DNA fragments will be cutting agent (such as the endonuclease) to carry DNA to a specific site, so as to achieve selective chromosome DNA hit off the end, because the cells Transcription factors such as regulation and protein only after the combination of DNA double helix to open its specific gene transcription, transcription factors can not helical and the three combined, it can make use of oligo DNA fragment closure of the transcription factor binding sites in order to reach the turn off harmful genes or Virus genes.
Quadruplex DNA
Klug and Sundpuist in a simulated 1 kind of spike protozoan telomere DNA of the caterpillar, paragraph 1 of the synthetic DNA sequences and found that under certain conditions, the simulation of G-rich single-stranded DNA to form a quadruplex DNA structure. This chromosome that the end of the telomere single strand between the formation of a four-chain. Kang and others were confirmed by experiments in crystal and in solution, the rich G DNA can form a quadruplex DNA structure.
Quadruplex DNA is the basic structure of the G-quadruplex, that is, in the four conjoined at the center there are 4 by a negative charge with the carboxylic oxygen atoms surrounded the "pocket" through the G-quadruplex accumulation can be Elements to form molecules or between right-handed spiral, with the double helix structure of DNA comparison, G-quadruplex spiral 2 significant characteristics: 1, the stability of its decision in the pocket by the combination of cation type, known ion k The combination of quadruple-helix so that the most stable; 2, and its thermodynamic stability of the very nature and dynamics.
At present some of the biological DNA sequence analysis that the G-rich DNA sequence found in some of the functions and evolution are very conservative region of the genome, many studies have shown that guanine-rich DNA chain formed by G-DNA may be As the mutual recognition between the molecular components of one of the organisms in the cell plays a special role
Thursday, October 15, 2009
DNA, RNA and Protein : Life at its simplest
DNA : Deoxyribonucleic acid. The double-stranded chemical instruction manual for everything a plant or animal does: grow, divide, even when and how to die. Very stable, has error detection and repair mechanisms. Stays in the cell nucleus. Can make good copies of itself.
RNA: Ribonucleic acid. Single-stranded where DNA is double-stranded, messenger RNA carries single pages of instructions out of the nucleus to places they're needed throughout the cell. No error detection or repair; makes flawed copies of itself. Evolves ten times faster than DNA. Transfer RNA helps translate the mRNA message into chains of amino acids in the ribosomes.
[Diagram of RNA vs. DNA: chemical structure and composition]
Base: a building block of DNA and RNA. There are five different bases: Adenine, Thymine, Guanine, Cytosine, and Uracil (which is found only in RNA and replaces Thymine in DNA).
Ribosomes: Message centers throughout the cell where the information from DNA arrives in the form of messenger RNA. The RNA message gets translated into a form the ribosome can understand and tells it which protein building blocks it needs and in what order to assemble them. Ribosomal RNA helps the translation go smoothly.
Amino acids: Polypeptide (protein) building blocks.
Polypeptides: chains of amino acids. Proteins are made up of several or many polypeptides.
Proteins: Chemicals that make up cell and organ structure and carry out reactions throughout the body, from breaking down food to fighting off disease.
RNA: Ribonucleic acid. Single-stranded where DNA is double-stranded, messenger RNA carries single pages of instructions out of the nucleus to places they're needed throughout the cell. No error detection or repair; makes flawed copies of itself. Evolves ten times faster than DNA. Transfer RNA helps translate the mRNA message into chains of amino acids in the ribosomes.
[Diagram of RNA vs. DNA: chemical structure and composition]
Base: a building block of DNA and RNA. There are five different bases: Adenine, Thymine, Guanine, Cytosine, and Uracil (which is found only in RNA and replaces Thymine in DNA).
Ribosomes: Message centers throughout the cell where the information from DNA arrives in the form of messenger RNA. The RNA message gets translated into a form the ribosome can understand and tells it which protein building blocks it needs and in what order to assemble them. Ribosomal RNA helps the translation go smoothly.
Amino acids: Polypeptide (protein) building blocks.
Polypeptides: chains of amino acids. Proteins are made up of several or many polypeptides.
Proteins: Chemicals that make up cell and organ structure and carry out reactions throughout the body, from breaking down food to fighting off disease.
Saturday, September 12, 2009
Technology of Genetic Engineering
Genetic engineering requires three fundamental technologies: the ability to isolate and modify the DNA of specific individual genes; an understanding of the mechanisms that regulate how genes function and how these can be manipulated; and the capacity to transfer genes into an organism. These have all been developed following the discovery of the structure of DNA in 1953. Genetic engineering of microbes was first reported in 1973, followed in the next decade by similar achievements in plants and animals. Because DNA is the genetic material in all organisms, genes for genetic engineering can be taken from any source, or even synthesized. Modification of genes may be necessary, particularly in regions that control how they operate, in order for the genes to function effectively in the recipient organism. Agrobacterium tumefaciens, a bacterium that transfers DNA into plant cells as part of its normal life cycle, is used commonly to transfer genes into plants, although other methods such as the "gene gun" also have been developed. Genetically engineered plants are technically "transgenic organisms," as they contain transferred genes. However, they are frequently referred to as "genetically modified organisms," or GMOs, and the products derived from them are described as "genetically modified," or GM foods. These terms can be confusing, as essentially all cultivated plants have been genetically modified through breeding and selection—for example, the many varieties of cultivated onions possess numerous qualities that distinguish them from each other and especially from the wild onions from which they originated.
Application of Genetic Engineering in Agriculture
The first genetically engineered crops were planted on a large scale in 1996. By 2001 more than fifty million hectares were planted worldwide with transgenic crops. The first generation of these crops has been altered in ways that improve the efficiency of crop production by modifying the tolerance of plants to herbicides and insect pests. Broad-spectrum herbicides are able to kill almost all plants. A prerequisite for using chemicals to control weeds in a crop is that the crop itself must be resistant to the herbicide. Genetic engineering has been used to develop plants (specifically soybean, canola, corn, and cotton) with resistance to two broad-spectrum herbicides, glyphosate and glufosinate, which are sold under the trademarks Roundup and Liberty, respectively. Glyphosate-tolerant soybeans have been adopted rapidly in some countries, notably the United States and Argentina, and accounted for approximately 46 percent of the soybean acreage worldwide in 2001. Herbicide use has not declined in these crops but the specific herbicides that are used have changed.
Insect pests can damage crops during the growing season and also after harvest. A variety of methods, including cultural practices and insecticides, are used to control insect damage. Genetic engineering has provided novel approaches to this problem. The bacterium Bacillus thuringiensis (Bt) produces proteins that are toxic to some types of insects, and Bt spores have been used as insecticides for decades. Genes encoding Bt toxin proteins have been isolated, modified so they function in plants, and transferred into crop plants including corn, potato, and cotton. These engineered Bt crops are more resistant to such insects as the European corn borer, Colorado potato beetle, and cotton bollworm than are their nonengineered counterparts. The introduction of Bt cotton has resulted in reduced use of insecticides on this crop in some regions of the United States. Growers of Bt crops are required to plant a portion of their acreage with varieties that do not carry the Bt gene, in an effort to delay the development of insect populations with resistance to Bt toxins.
The Flavr Savr tomato, developed in the 1980s by Calgene, a biotechnology company in California, was the first food produced from a genetically engineered plant. These tomatoes ripened more slowly and had an extended shelf life. However, for a number of reasons—including production problems and consumer skepticism—this product was not a commercial success and was withdrawn in 1996, after less than three years on the market. Melons and raspberries have also been engineered to have delayed ripening but have not been produced commercially. Transgenic papayas with resistance to ring spot virus also have been developed. These were grown successfully in Hawaii, where the papaya industry was devastated by this debilitating disease. A similar approach was used to produce virus-resistant summer squash and against other viruses affecting a wide variety of foodstuffs.
The first generation of transgenic crops for the most part were designed to improve the efficiency of crop production, an ongoing objective for genetic engineers. Additionally, the techniques of genetic engineering can be used to alter the nutritional composition of foods. The transfer into rice of three genes that function to produce beta-carotene in the seed resulted in "golden rice." Once consumed, beta-carotene can be converted to vitamin A, the degree of this conversion being dependent upon a number of factors that relate to the source of the beta-carotene, the diet, and the individual consumer. In less-developed countries, vitamin A deficiency is widespread among those with a restricted diet, and is responsible for increased mortality and blindness in children. Although the efficacy of transgenic rice in reducing disease has not been established, it demonstrates the potential use of genetic engineering for nutritional enhancement in many crops. Other applications of genetic engineering of animal and human foods include removing allergens from foods such as peanuts, increasing the level of essential vitamins and nutrients in foods, and producing foods possessed of vaccines and other beneficial compounds.
Genetically engineered microbes also are used to produce proteins for food processing. Chymosin (or rennin), an enzyme used in cheese production, traditionally is obtained from the stomach of veal calves. However, the gene encoding this enzyme was transferred into microbes, and the enzyme now can be produced in bulk by purifying it from large microbe cultures. Chymosin prepared from transgenic microbes has more predictable properties than the animal product and is used to produce more than fifty percent of hard cheeses in the United States. Other enzymes used in food processing are produced by similar methods. For example, bovine growth hormone (BGH) is produced in large quantities from transgenic microbes and is given to cows to increase milk production.
Application of Genetic Engineering in Agriculture
The first genetically engineered crops were planted on a large scale in 1996. By 2001 more than fifty million hectares were planted worldwide with transgenic crops. The first generation of these crops has been altered in ways that improve the efficiency of crop production by modifying the tolerance of plants to herbicides and insect pests. Broad-spectrum herbicides are able to kill almost all plants. A prerequisite for using chemicals to control weeds in a crop is that the crop itself must be resistant to the herbicide. Genetic engineering has been used to develop plants (specifically soybean, canola, corn, and cotton) with resistance to two broad-spectrum herbicides, glyphosate and glufosinate, which are sold under the trademarks Roundup and Liberty, respectively. Glyphosate-tolerant soybeans have been adopted rapidly in some countries, notably the United States and Argentina, and accounted for approximately 46 percent of the soybean acreage worldwide in 2001. Herbicide use has not declined in these crops but the specific herbicides that are used have changed.
Insect pests can damage crops during the growing season and also after harvest. A variety of methods, including cultural practices and insecticides, are used to control insect damage. Genetic engineering has provided novel approaches to this problem. The bacterium Bacillus thuringiensis (Bt) produces proteins that are toxic to some types of insects, and Bt spores have been used as insecticides for decades. Genes encoding Bt toxin proteins have been isolated, modified so they function in plants, and transferred into crop plants including corn, potato, and cotton. These engineered Bt crops are more resistant to such insects as the European corn borer, Colorado potato beetle, and cotton bollworm than are their nonengineered counterparts. The introduction of Bt cotton has resulted in reduced use of insecticides on this crop in some regions of the United States. Growers of Bt crops are required to plant a portion of their acreage with varieties that do not carry the Bt gene, in an effort to delay the development of insect populations with resistance to Bt toxins.
The Flavr Savr tomato, developed in the 1980s by Calgene, a biotechnology company in California, was the first food produced from a genetically engineered plant. These tomatoes ripened more slowly and had an extended shelf life. However, for a number of reasons—including production problems and consumer skepticism—this product was not a commercial success and was withdrawn in 1996, after less than three years on the market. Melons and raspberries have also been engineered to have delayed ripening but have not been produced commercially. Transgenic papayas with resistance to ring spot virus also have been developed. These were grown successfully in Hawaii, where the papaya industry was devastated by this debilitating disease. A similar approach was used to produce virus-resistant summer squash and against other viruses affecting a wide variety of foodstuffs.
The first generation of transgenic crops for the most part were designed to improve the efficiency of crop production, an ongoing objective for genetic engineers. Additionally, the techniques of genetic engineering can be used to alter the nutritional composition of foods. The transfer into rice of three genes that function to produce beta-carotene in the seed resulted in "golden rice." Once consumed, beta-carotene can be converted to vitamin A, the degree of this conversion being dependent upon a number of factors that relate to the source of the beta-carotene, the diet, and the individual consumer. In less-developed countries, vitamin A deficiency is widespread among those with a restricted diet, and is responsible for increased mortality and blindness in children. Although the efficacy of transgenic rice in reducing disease has not been established, it demonstrates the potential use of genetic engineering for nutritional enhancement in many crops. Other applications of genetic engineering of animal and human foods include removing allergens from foods such as peanuts, increasing the level of essential vitamins and nutrients in foods, and producing foods possessed of vaccines and other beneficial compounds.
Genetically engineered microbes also are used to produce proteins for food processing. Chymosin (or rennin), an enzyme used in cheese production, traditionally is obtained from the stomach of veal calves. However, the gene encoding this enzyme was transferred into microbes, and the enzyme now can be produced in bulk by purifying it from large microbe cultures. Chymosin prepared from transgenic microbes has more predictable properties than the animal product and is used to produce more than fifty percent of hard cheeses in the United States. Other enzymes used in food processing are produced by similar methods. For example, bovine growth hormone (BGH) is produced in large quantities from transgenic microbes and is given to cows to increase milk production.
Saturday, August 15, 2009
The History of Genetic Engineering
Genetic engineering owes its existence to the developments in molecular genetics, virology, and cytology that culminated in the determination of the structure of DNA by James Watson and Francis Crick in 1953. Building on research involving bacteriophages (a bacterial virus), Joshua Lederberg, a geneticist at the University of Wisconsin, found that bacteria can transfer genetic information through plasmids, small mobile pieces of DNA that exist independent of the chromosomes. In the 1950s, Lederberg pioneered the earliest techniques in genetic engineering, shuffling genetic material between bacterial cells. After the identification of restriction enzymes capable of "cutting" DNA in specific locations in 1968, scientists were able to insert foreign DNA directly into bacterial cells. The discovery that the foreign DNA would naturally bond with the host DNA, made it possible to splice together genes from multiple organisms, the technique used in recombinant DNA engineering. Although highly complicated, rDNA engineering can be simply explained: genetic material from the donor source is isolated and "cut" using a restriction enzyme and then recombined or "pasted" into the genetic material of the receiver. By 1971, advanced transplantation techniques had been developed and rDNA techniques using the restriction enzyme EcoRi were operable the following year, leading to the first experiments in genetic engineering.
In 1973, Stanford biochemist Stanley Cohen under-took one of the first rDNA experiments, inserting a piece of bacterial DNA into Escherichia coli (E. coli), a bacterium found in the human intestine. However, the research soon became controversial, particularly when American molecular biologist Paul Berg designed an experiment to insert DNA from simian virus #40 (sv40)—a known cancer-causing agent—into E. coli. As word of the daring procedure spread, the public was captivated and fearful, afraid that a genetically engineered virus, inured to antibiotics and carried in a common bacterium, could escape and cause an epidemic. Hoping to diffuse fears of a potential biohazard and maintain control of their research, over one hundred and fifty molecular biologists and related specialists met at the Asilomar Conference Center in Monterey, California, in late February 1975. The conference represented an extraordinary moment in the history of science, as the research community, recognizing its social responsibility, officially adopted a moratorium until appropriately safe procedures and guidelines could be developed. The conference ultimately resulted in the "National Institutes of Health Guidelines for Research Involving rDNA Molecules" and an ongoing National Institute of Health rDNA Advisory Committee (RAC)founded in 1974.
Yet the guidelines only increased public concern over genetic engineering. Critics charged that attempts to splice genes together from different organisms were akin to "playing God" and could result in dangerous and immoral hybrids. Adopting the literary example of "Dr. Frankenstein's monster" as an appropriate symbol of misguided science, opponents of rDNA engineering converged on research laboratories and public meetings. An attempt to build a recombinant laboratory at Harvard University set off such a firestorm that local politicians created a review board to assess potential risks, eventually requiring more stringent controls than those set by the NIH. By 1977, protests of rDNA facilities had spread to other campuses—the University of California San Diego, the University of Wisconsin, the University of Michigan, and the University of Indiana—while the state legislatures of New York, New Jersey, and California held public hearings. However, it was the resolution of an old court case and the introduction of a new form of rDNA engineering that ultimately created the greatest controversy.
In a monumental decision handed down on 16 June 1980, the United States Supreme Court held in Diamond v. Chakrabarty that man-made life forms were subject to patent laws and protection. The decision resolved a longstanding issue on patents and organic material, as the case dated to 1972, when Ananda Chakrabarty, a researcher at General Electric, applied for a patent on a form of Pseudomonas bacteria bred (but not genetically engineered)to digest oil slicks. By a narrow five to four margin the court construed the Patent Act, originally drafted by Thomas Jefferson, so as to include all products of human invention, relying on a 1952 Senate report that recognized as patentable "anything under the sun that is made by man." More than any other single event, the ruling galvanized many mainstream religious communities and environmental groups, eventually resulting in a letter of protest to President Carter and an indepth review by the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research (1980–1983). The commission's report, issued in 1982 and entitled Splicing Life: The Social and Ethical Issues of Genetic Engineering with Human Beings, emphasized the importance of rDNA engineering to biomedical progress and American industries, arguing that it was best that the research be conducted under the auspices of government regulation and control. However, while the study resolved anxiety over rDNA engineering and patenting, proponents of genetic engineering still had to address concerns over the development of "germ-line" engineering, a controversial procedure that allowed scientists to literally create new strains of organisms.
Germ-line engineering differs from rDNA engineering in that the donor genes are inserted into a "germ," or reproductive cell, thereby permanently altering the genetic makeup of the organism's descendants. For example, in 1982, Ralph Brinster of the University of Pennsylvania Veterinary School inserted the gene that produces rat growth hormone into mouse embryos. The resulting strain of mice, dubbed "super mice" by the press, expressed the gene and thus grew into a substantially larger and more powerful new breed of mouse. Critics of germ-line engineering quickly denounced the technique as immoral and argued it was a form of "anthropomorphic Lamarckism."
Jean-Baptiste de Lamarck, a nineteenth-century French naturalist, had proposed that traits acquired during an organism's lifetime were passed on to its progeny—an idea refuted by Darwinian evolutionary theory. Yet, in germ-line engineering, traits acquired during the organism's lifetime are passed on, but only those traits deemed necessary or desirous by man. Environmental groups also denounced germ-line engineering because of "biosafety" concerns, fearing that genetically engineered species, which would possess a distinct advantage over nonengineered species, could upset the globe's finely tuned ecological systems. However, because most politicians, scientists, and manufacturers believed the potential benefits from rDNA and germ-line engineering outweighed its potential dangers, the protests were overshadowed by the development of a biotechnology industry based on genetic engineering.
In 1973, Stanford biochemist Stanley Cohen under-took one of the first rDNA experiments, inserting a piece of bacterial DNA into Escherichia coli (E. coli), a bacterium found in the human intestine. However, the research soon became controversial, particularly when American molecular biologist Paul Berg designed an experiment to insert DNA from simian virus #40 (sv40)—a known cancer-causing agent—into E. coli. As word of the daring procedure spread, the public was captivated and fearful, afraid that a genetically engineered virus, inured to antibiotics and carried in a common bacterium, could escape and cause an epidemic. Hoping to diffuse fears of a potential biohazard and maintain control of their research, over one hundred and fifty molecular biologists and related specialists met at the Asilomar Conference Center in Monterey, California, in late February 1975. The conference represented an extraordinary moment in the history of science, as the research community, recognizing its social responsibility, officially adopted a moratorium until appropriately safe procedures and guidelines could be developed. The conference ultimately resulted in the "National Institutes of Health Guidelines for Research Involving rDNA Molecules" and an ongoing National Institute of Health rDNA Advisory Committee (RAC)founded in 1974.
Yet the guidelines only increased public concern over genetic engineering. Critics charged that attempts to splice genes together from different organisms were akin to "playing God" and could result in dangerous and immoral hybrids. Adopting the literary example of "Dr. Frankenstein's monster" as an appropriate symbol of misguided science, opponents of rDNA engineering converged on research laboratories and public meetings. An attempt to build a recombinant laboratory at Harvard University set off such a firestorm that local politicians created a review board to assess potential risks, eventually requiring more stringent controls than those set by the NIH. By 1977, protests of rDNA facilities had spread to other campuses—the University of California San Diego, the University of Wisconsin, the University of Michigan, and the University of Indiana—while the state legislatures of New York, New Jersey, and California held public hearings. However, it was the resolution of an old court case and the introduction of a new form of rDNA engineering that ultimately created the greatest controversy.
In a monumental decision handed down on 16 June 1980, the United States Supreme Court held in Diamond v. Chakrabarty that man-made life forms were subject to patent laws and protection. The decision resolved a longstanding issue on patents and organic material, as the case dated to 1972, when Ananda Chakrabarty, a researcher at General Electric, applied for a patent on a form of Pseudomonas bacteria bred (but not genetically engineered)to digest oil slicks. By a narrow five to four margin the court construed the Patent Act, originally drafted by Thomas Jefferson, so as to include all products of human invention, relying on a 1952 Senate report that recognized as patentable "anything under the sun that is made by man." More than any other single event, the ruling galvanized many mainstream religious communities and environmental groups, eventually resulting in a letter of protest to President Carter and an indepth review by the President's Commission for the Study of Ethical Problems in Medicine and Biomedical and Behavioral Research (1980–1983). The commission's report, issued in 1982 and entitled Splicing Life: The Social and Ethical Issues of Genetic Engineering with Human Beings, emphasized the importance of rDNA engineering to biomedical progress and American industries, arguing that it was best that the research be conducted under the auspices of government regulation and control. However, while the study resolved anxiety over rDNA engineering and patenting, proponents of genetic engineering still had to address concerns over the development of "germ-line" engineering, a controversial procedure that allowed scientists to literally create new strains of organisms.
Germ-line engineering differs from rDNA engineering in that the donor genes are inserted into a "germ," or reproductive cell, thereby permanently altering the genetic makeup of the organism's descendants. For example, in 1982, Ralph Brinster of the University of Pennsylvania Veterinary School inserted the gene that produces rat growth hormone into mouse embryos. The resulting strain of mice, dubbed "super mice" by the press, expressed the gene and thus grew into a substantially larger and more powerful new breed of mouse. Critics of germ-line engineering quickly denounced the technique as immoral and argued it was a form of "anthropomorphic Lamarckism."
Jean-Baptiste de Lamarck, a nineteenth-century French naturalist, had proposed that traits acquired during an organism's lifetime were passed on to its progeny—an idea refuted by Darwinian evolutionary theory. Yet, in germ-line engineering, traits acquired during the organism's lifetime are passed on, but only those traits deemed necessary or desirous by man. Environmental groups also denounced germ-line engineering because of "biosafety" concerns, fearing that genetically engineered species, which would possess a distinct advantage over nonengineered species, could upset the globe's finely tuned ecological systems. However, because most politicians, scientists, and manufacturers believed the potential benefits from rDNA and germ-line engineering outweighed its potential dangers, the protests were overshadowed by the development of a biotechnology industry based on genetic engineering.
Wednesday, August 5, 2009
Animal cells and plant cells
Animal cells
In contrast to the wide variety of plasmid and phage vectors available for cloning in prokaryotic cells, relatively few vectors are available for introducing foreign genes into animal cells. The most commonly used are derived from simian virus 40 (SV40). Normal SV40 cannot be used as a vector, since there is a physical limit to the amount of DNA that can be packaged into the virus capsid, and the addition of foreign DNA would generate a DNA molecule too large to be packaged. However, SV40 mutants lacking portions of the genome can be propagated in mixed infections in which a “helper” virus supplies the missing function.
Plant cells
Two systems for the delivery and integration of foreign genes into the plant genome are the Ti plasmid of the soil bacterium Agrobacterium and the DNA plant virion cauliflower mosaic virus. The Ti plasmid is a natural gene transfer vector carried by A. tumefaciens, a pathogenic bacterium that causes crown gall tumor formation in dicotyledonous plants. A T-DNA segment present in the Ti plasmid becomes stably integrated into the plant cell genome during infection. This property of the Ti plasmid has been exploited to show that DNA segments inserted in the T-DNA region can be cotransferred to plant DNA.
Applications
Recombinant DNA technology has permitted the isolation and detailed structural analysis of a large number of prokaryotic and eukaryotic genes. This contribution is especially significant in the eukaryotes because of their large genomes. The methods outlined above provide a means of fractionating and isolating individual genes, since each clone contains a single sequence or a few DNA sequences from a very large genome. Isolation of a particular sequence of interest has been facilitated by the ability to generate a large number of clones and to screen them with the appropriate “probe” (radioactively labeled RNA or DNA) molecules.
Genetic engineering techniques provide pure DNAs in amounts sufficient for mapping, sequencing, and direct structural analyses. Furthermore, gene structure-function relationships can be studied by reintroducing the cloned gene into a eukaryotic nucleus and assaying for transcriptional and translational activities. The DNA sequences can be altered by mutagenesis before their reintroduction in order to define precise functional regions.
Genetic engineering methodology has provided means for the large-scale production of polypeptides and proteins. It is now possible to produce a wide variety of foreign proteins in E. coli. These range from enzymes useful in molecular biology to a vast range of polypeptides with potential human therapeutic applications, such as insulin, interferon, growth hormone, immunoglobins, and enzymes involved in the dynamics of blood coagulation.
In contrast to the wide variety of plasmid and phage vectors available for cloning in prokaryotic cells, relatively few vectors are available for introducing foreign genes into animal cells. The most commonly used are derived from simian virus 40 (SV40). Normal SV40 cannot be used as a vector, since there is a physical limit to the amount of DNA that can be packaged into the virus capsid, and the addition of foreign DNA would generate a DNA molecule too large to be packaged. However, SV40 mutants lacking portions of the genome can be propagated in mixed infections in which a “helper” virus supplies the missing function.
Plant cells
Two systems for the delivery and integration of foreign genes into the plant genome are the Ti plasmid of the soil bacterium Agrobacterium and the DNA plant virion cauliflower mosaic virus. The Ti plasmid is a natural gene transfer vector carried by A. tumefaciens, a pathogenic bacterium that causes crown gall tumor formation in dicotyledonous plants. A T-DNA segment present in the Ti plasmid becomes stably integrated into the plant cell genome during infection. This property of the Ti plasmid has been exploited to show that DNA segments inserted in the T-DNA region can be cotransferred to plant DNA.
Applications
Recombinant DNA technology has permitted the isolation and detailed structural analysis of a large number of prokaryotic and eukaryotic genes. This contribution is especially significant in the eukaryotes because of their large genomes. The methods outlined above provide a means of fractionating and isolating individual genes, since each clone contains a single sequence or a few DNA sequences from a very large genome. Isolation of a particular sequence of interest has been facilitated by the ability to generate a large number of clones and to screen them with the appropriate “probe” (radioactively labeled RNA or DNA) molecules.
Genetic engineering techniques provide pure DNAs in amounts sufficient for mapping, sequencing, and direct structural analyses. Furthermore, gene structure-function relationships can be studied by reintroducing the cloned gene into a eukaryotic nucleus and assaying for transcriptional and translational activities. The DNA sequences can be altered by mutagenesis before their reintroduction in order to define precise functional regions.
Genetic engineering methodology has provided means for the large-scale production of polypeptides and proteins. It is now possible to produce a wide variety of foreign proteins in E. coli. These range from enzymes useful in molecular biology to a vast range of polypeptides with potential human therapeutic applications, such as insulin, interferon, growth hormone, immunoglobins, and enzymes involved in the dynamics of blood coagulation.
Tuesday, July 28, 2009
Cloning vectors
There is a large variety of potential vectors for cloned genes. The vectors differ in different classes of organisms.
Prokaryotes and lower eukaryotes
Three types of vectors have been used in these organisms: plasmids, bacteriophages, and cosmids. Plasmids are extrachromosomal DNA sequences that are stably inherited. Escherichia coli and its plasmids constitute the most versatile type of host-vector system known for DNA cloning. Several natural plasmids, such as ColE1, have been used as cloning vehicles in E. coli. In addition, a variety of derivatives of natural plasmids have been constructed by combining DNA segments and desirable qualities of older cloning vehicles. The most versatile and widely used of these plasmids is pBR322. Transformation in yeast has been demonstrated using a number of plasmids, including vectors derived from the naturally occurring 2μ plasmid of yeast.
Bacteriophage lambda is a virus of E. coli. Several lambda-derived vectors have been developed for cloning in E. coli, and for the isolation of particular genes from eukaryotic genomes. These lambda derivatives have several advantages over plasmids: (1) Thousands of recombinant phage plaques can easily be screened for a particular DNA sequence on a single petri dish by molecular hybridization. (2) Packaging of recombinant DNA in laboratory cultures provides a very efficient means of DNA uptake by the bacteria. (3) Thousands of independently packaged recombinant phages can be easily replicated and stored in a single solution as a “library” of genomic sequences.
Plasmids have also been constructed that contain the phage cos DNA site, required for packaging into the phage particles, and ColE1 DNA segments, required for plasmid replication. These plasmids have been termed cosmids. The recombinant cosmid DNA is injected into a host and circularizes like phage DNA but replicates as a plasmid. Transformed cells are selected on the basis of a vector drug resistance marker.
Prokaryotes and lower eukaryotes
Three types of vectors have been used in these organisms: plasmids, bacteriophages, and cosmids. Plasmids are extrachromosomal DNA sequences that are stably inherited. Escherichia coli and its plasmids constitute the most versatile type of host-vector system known for DNA cloning. Several natural plasmids, such as ColE1, have been used as cloning vehicles in E. coli. In addition, a variety of derivatives of natural plasmids have been constructed by combining DNA segments and desirable qualities of older cloning vehicles. The most versatile and widely used of these plasmids is pBR322. Transformation in yeast has been demonstrated using a number of plasmids, including vectors derived from the naturally occurring 2μ plasmid of yeast.
Bacteriophage lambda is a virus of E. coli. Several lambda-derived vectors have been developed for cloning in E. coli, and for the isolation of particular genes from eukaryotic genomes. These lambda derivatives have several advantages over plasmids: (1) Thousands of recombinant phage plaques can easily be screened for a particular DNA sequence on a single petri dish by molecular hybridization. (2) Packaging of recombinant DNA in laboratory cultures provides a very efficient means of DNA uptake by the bacteria. (3) Thousands of independently packaged recombinant phages can be easily replicated and stored in a single solution as a “library” of genomic sequences.
Plasmids have also been constructed that contain the phage cos DNA site, required for packaging into the phage particles, and ColE1 DNA segments, required for plasmid replication. These plasmids have been termed cosmids. The recombinant cosmid DNA is injected into a host and circularizes like phage DNA but replicates as a plasmid. Transformed cells are selected on the basis of a vector drug resistance marker.
Saturday, July 25, 2009
Joining DNA molecules
Once the proper DNA fragments have been obtained, they must be joined. When cleavage with a restriction endonuclease creates cohesive ends, these can be annealed with a similarly cleaved DNA from another source, including a vector molecule. When such molecules associate, the joint has nicks a few base pairs apart in opposite strands. The enzyme DNA ligase can then repair these nicks to form an intact, duplex recombinant molecule, which can be used for transformation and the subsequent selection of cells containing the recombinant molecule. Cohesive ends can also be created by the addition of synthetic DNA linkers to blunt-ended DNA molecules.
Another method for joining DNA molecules involves the addition of homopolymer extensions to different DNA populations followed by an annealing of complementary homopolymer sequences. For example, short nucleotide sequences of pure adenine can be added to the 3′ ends of one population of DNA molecules and short thymine blocks to the 3′ ends of another population. The two types of molecules can then anneal to form mixed dimeric circles that can be used directly for transformation.
The enzyme T4 DNA ligase carries out the intermolecular joining of DNA substrates at completely base-paired ends; such blunt ends can be produced by cleavage with a restriction enzyme or by mechanical shearing followed by enzyme treatment.
Transformation
The desired DNA sequence, once attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is usually referred to as transferring.
Transformation in any organism involves (1) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity. See also Transformation (bacteria).
Escherichia coli is usually the host of choice for cloning experiments, and transformation of E. coli is an essential step in these experiments. Escherichia coli treated with calcium chloride are able to take up DNA from bacteriophage lambda as well as plasmid DNA. Calcium chloride is thought to effect some structural alterations in the bacterial cell wall. An efficient method for transformation in Bacillus species involves polyethylene glycol-induced DNA uptake in bacterial protoplasts and subsequent regeneration of the bacterial cell wall. Actinomycetes can be similarly transformed. Transformation can also be achieved by first entrapping the DNA with liposomes followed by their fusion with the host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa.
Another method for joining DNA molecules involves the addition of homopolymer extensions to different DNA populations followed by an annealing of complementary homopolymer sequences. For example, short nucleotide sequences of pure adenine can be added to the 3′ ends of one population of DNA molecules and short thymine blocks to the 3′ ends of another population. The two types of molecules can then anneal to form mixed dimeric circles that can be used directly for transformation.
The enzyme T4 DNA ligase carries out the intermolecular joining of DNA substrates at completely base-paired ends; such blunt ends can be produced by cleavage with a restriction enzyme or by mechanical shearing followed by enzyme treatment.
Transformation
The desired DNA sequence, once attached to a DNA vector, must be transferred to a suitable host. Transformation is defined as the introduction of foreign DNA into a recipient cell. Transformation of a cell with DNA from a virus is usually referred to as transferring.
Transformation in any organism involves (1) a method that allows the introduction of DNA into the cell and (2) the stable integration of DNA into a chromosome, or maintenance of the DNA as a self-replicating entity. See also Transformation (bacteria).
Escherichia coli is usually the host of choice for cloning experiments, and transformation of E. coli is an essential step in these experiments. Escherichia coli treated with calcium chloride are able to take up DNA from bacteriophage lambda as well as plasmid DNA. Calcium chloride is thought to effect some structural alterations in the bacterial cell wall. An efficient method for transformation in Bacillus species involves polyethylene glycol-induced DNA uptake in bacterial protoplasts and subsequent regeneration of the bacterial cell wall. Actinomycetes can be similarly transformed. Transformation can also be achieved by first entrapping the DNA with liposomes followed by their fusion with the host cell membrane. Similar transformation methods have been developed for lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Neurospora crassa.
Wednesday, July 15, 2009
Isolation of passenger DNA
Passenger DNA may be isolated in a number of ways; the most common of these involves DNA restriction. Restriction endonucleases make possible the cleavage of high-molecular-weight DNA. Although three different classes of these enzymes have been described, only type II restriction endonucleases have been used extensively in the manipulation of DNA. Type II restriction endonucleases are DNAases that recognize specific short nucleotide sequences (usually 4 to 6 base pairs in length), and then cleave both strands of the DNA duplex, generating discrete DNA fragments of defined length and sequence. A number of restriction enzymes make staggered cuts in the two DNA strands, generating single-stranded termini. See also Restriction enzyme.
The various fragments generated when a specific DNA is cut by a restriction enzyme can be easily resolved as bands of distinct molecular weights by agarose gel electrophoresis. Specific sequences of these bands can be identified by a technique known as Southern blotting. In this technique, DNA restriction fragments resolved on a gel are denatured and blotted onto a nitrocellulose filter. The filter is incubated together with a radioactively labeled DNA or RNA probe specific for the gene under study. The labeled probe hybridizes to its complement in the restricted DNA, and the regions of hybridization are detected autoradiographically. Fragments of interest can then be eluted out of these gels and used for cloning. Purification of particular DNA segments prior to cloning reduces the number of recombinant that must later be screened. See also Electrophoresis.
Another method that has been used to generate small DNA fragments is mechanical shearing. Intense personification of high-molecular-weight DNA with ultrasound, or high-speed stirring in a blender, can both be used to produce DNA fragments of a certain size range. Shearing results in random breakage of DNA, producing termini consisting of short, single-stranded regions. Other sources include DNA complementary to poly(A) RNA, or cDNA, which is synthesized in the test tube, and short oligonucleotides that are synthesized chemically.
The various fragments generated when a specific DNA is cut by a restriction enzyme can be easily resolved as bands of distinct molecular weights by agarose gel electrophoresis. Specific sequences of these bands can be identified by a technique known as Southern blotting. In this technique, DNA restriction fragments resolved on a gel are denatured and blotted onto a nitrocellulose filter. The filter is incubated together with a radioactively labeled DNA or RNA probe specific for the gene under study. The labeled probe hybridizes to its complement in the restricted DNA, and the regions of hybridization are detected autoradiographically. Fragments of interest can then be eluted out of these gels and used for cloning. Purification of particular DNA segments prior to cloning reduces the number of recombinant that must later be screened. See also Electrophoresis.
Another method that has been used to generate small DNA fragments is mechanical shearing. Intense personification of high-molecular-weight DNA with ultrasound, or high-speed stirring in a blender, can both be used to produce DNA fragments of a certain size range. Shearing results in random breakage of DNA, producing termini consisting of short, single-stranded regions. Other sources include DNA complementary to poly(A) RNA, or cDNA, which is synthesized in the test tube, and short oligonucleotides that are synthesized chemically.
Friday, July 10, 2009
Cloning of cells
A clone is a cell, group of cells, or organism that contains genetic information identical to that of the parent cell or organism. It is a form of asexual reproduction (see Reproduction), and as such it is not as new as it seems; what is new, however, is humans' ability to manipulate cloning at the genetic level. The first clones produced by humans as long as 2,000 years ago were plants developed from grafts and stem cuttings. By cloning—a process that calls into play complex laboratory techniques and the use of DNA replication—people usually mean a relatively recent scientific advance. Among these techniques is the ability to isolate and copy (that is, to clone) individual genes that direct an organism's development.
The Promise of Cloning
The cloning of specific genes can provide large numbers of copies of that gene for use in genetic and taxonomic research as well as in the practical areas of medicine and farming. In the latter field, the goal is to clone plants with specific traits that make them superior to naturally occurring organisms. For example, in 1985 scientists conducted field tests using clones of plants whose genes had been altered in the laboratory to generate resistance to insects, viruses, and bacteria. New strains of plants resulting from cloning could produce crops that can grow in poor soil or even underwater and fruits and vegetables with improved nutritional qualities and longer shelf lives. A cloning technique known as twinning could induce livestock to give birth to twins or even triplets, and on the environmental front cloning might help save endangered species from extinction.
In the realm of medicine and health, cloning has been used to make vaccines and hormones. It has become possible, by combining two different kinds of cells (such as mouse and human cancer cells), to produce large quantities of specific antibodies, via the immune system, to fight off disease. When injected into the bloodstream, these cloned antibodies seek out and attack disease-causing cells anywhere in the body. By attaching a tracer element to the cloned antibodies, scientists can locate hidden cancers, and by attaching specific cancer-fighting drugs, the treatment dose can be transported directly to the cancer cells.
Experiments in Cloning
The modern era of laboratory cloning began in 1958 when the British plant physiologist F. C. Steward (1904-1993) cloned carrot plants from mature single cells placed in a nutrient culture containing hormones. The first cloning of animal cells took place in 1964, when the British molecular biologist John B. Gurdon (1933-1989) took nuclei from intestinal cells of toad tadpoles and injected them into unfertilized eggs. The cell nuclei in the eggs had been destroyed with ultra-violet light, but when the eggs were incubated, Gurdon found that 1-2% of the eggs developed into fertile, adult toads.
The first successful cloning of mammals occurred nearly 20 years later, when scientists in Switzerland and the United States successfully cloned mice using a method similar to Gurdon's approach. Their method required one extra step, however: after taking the nuclei from the embryos of one type of mouse, they transferred them into the embryos of another type of mouse. The latter served as a surrogate, or replacement, mother. The cloning of cattle livestock was tried first in 1988, when embryos from prize cows were transplanted to unfertilized cow eggs whose own nuclei had been removed. An even greater breakthrough transpired on February 24, 1997, with the birth of a lamb named Dolly in Edinburgh, Scotland. Dolly was no ordinary sheep: she was the first mammal born from the cloning of an adult cell. Thus, she had been produced by asexual reproduction in the form of genetically engineered cloning rather than by anything resembling a normal process. Nonetheless, she proved her own ability to reproduce the old-fashioned way when, on April 23, 1998, she gave birth to a daughter named Bonnie.
The Promise of Cloning
The cloning of specific genes can provide large numbers of copies of that gene for use in genetic and taxonomic research as well as in the practical areas of medicine and farming. In the latter field, the goal is to clone plants with specific traits that make them superior to naturally occurring organisms. For example, in 1985 scientists conducted field tests using clones of plants whose genes had been altered in the laboratory to generate resistance to insects, viruses, and bacteria. New strains of plants resulting from cloning could produce crops that can grow in poor soil or even underwater and fruits and vegetables with improved nutritional qualities and longer shelf lives. A cloning technique known as twinning could induce livestock to give birth to twins or even triplets, and on the environmental front cloning might help save endangered species from extinction.
In the realm of medicine and health, cloning has been used to make vaccines and hormones. It has become possible, by combining two different kinds of cells (such as mouse and human cancer cells), to produce large quantities of specific antibodies, via the immune system, to fight off disease. When injected into the bloodstream, these cloned antibodies seek out and attack disease-causing cells anywhere in the body. By attaching a tracer element to the cloned antibodies, scientists can locate hidden cancers, and by attaching specific cancer-fighting drugs, the treatment dose can be transported directly to the cancer cells.
Experiments in Cloning
The modern era of laboratory cloning began in 1958 when the British plant physiologist F. C. Steward (1904-1993) cloned carrot plants from mature single cells placed in a nutrient culture containing hormones. The first cloning of animal cells took place in 1964, when the British molecular biologist John B. Gurdon (1933-1989) took nuclei from intestinal cells of toad tadpoles and injected them into unfertilized eggs. The cell nuclei in the eggs had been destroyed with ultra-violet light, but when the eggs were incubated, Gurdon found that 1-2% of the eggs developed into fertile, adult toads.
The first successful cloning of mammals occurred nearly 20 years later, when scientists in Switzerland and the United States successfully cloned mice using a method similar to Gurdon's approach. Their method required one extra step, however: after taking the nuclei from the embryos of one type of mouse, they transferred them into the embryos of another type of mouse. The latter served as a surrogate, or replacement, mother. The cloning of cattle livestock was tried first in 1988, when embryos from prize cows were transplanted to unfertilized cow eggs whose own nuclei had been removed. An even greater breakthrough transpired on February 24, 1997, with the birth of a lamb named Dolly in Edinburgh, Scotland. Dolly was no ordinary sheep: she was the first mammal born from the cloning of an adult cell. Thus, she had been produced by asexual reproduction in the form of genetically engineered cloning rather than by anything resembling a normal process. Nonetheless, she proved her own ability to reproduce the old-fashioned way when, on April 23, 1998, she gave birth to a daughter named Bonnie.
Sunday, July 5, 2009
DNA Big real life application
Ever since the breakthrough discoveries of Watson, Crick, and others in the 1950s made genetic engineering a possibility, the new field has promised increasingly bigger payoffs. These payoffs take the form of improvements to human life and profits to those who facilitate those improvements. The possible applications of genetic engineering are virtually limitless—as are the profits to be made from genetic engineering as a business. As early as the 1970s, entrepreneurs (independent businesspeople) recognized the commercial potential of genetically engineered products, which promised to revolutionize life, technology, and commerce as computers also were doing. Thus was born one of the great buzzwords of the late twentieth century: biotechnology, or the use of genetic engineering for commercial purposes.
Several early biotechnology firms were founded by scientists involved in fundamental research: Boyer, for example, teamed up with the venture capitalist Robert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other pioneering companies, including Cetus, Biogen, and Genex, likewise were founded through the collaboration of scientists and businesspeople. Today biotechnology promises a revolution in numerous areas, such as agriculture. Recombinant DNA techniques enable scientists to produce plants that are resistant to freezing temperatures, that will take longer to ripen, that will develop their own resistance to pests, and so on. By 1988 scientists had tested more than two dozen kinds of plants engineered to have special properties such as these. Yet no field of biotechnology and genetic engineering is as significant as the applications to health and the cures for diseases.
Medicines and Cures
The use of rDNA allows scientists to produce many products that were previously available only in limited quantities: for example, insulin, which we referred to earlier. Until the 1980s the only source of insulin for people with diabetes came from animals slaughtered for meat and other purposes. The supply was never high enough to meet demand, and this drove up prices. Then, in 1982, the U.S. Food and Drug Administration (FDA) approved the sale of insulin produced by genetically altered organisms—the first such product to become available. Since 1982 several additional products, such as human growth hormone, have been made with rDNA techniques.
One of the most exciting potential applications of genetic engineering is the treatment of genetic disorders, which are discussed in Heredity, through the use of gene therapy. Among the more than 3,000 such disorders, quite a few of which are quite serious or even fatal, many are the result of relatively minor errors in DNA sequencing. Genetic engineering offers the potential to provide individuals with correct copies of a gene, which could make possible a cure for that condition. In the 1980s scientists began clinical trials of a procedure known as human gene therapy to replace defective genes. The technique, still very much in the developmental stage, offers the hope of cures for diseases that medicine has long been powerless to combat.
In 2001 scientists at the Weizmann Institute in Israel brought together two of the most exciting fields of research, biotechnology and computers, to produce the DNA-processing nanocomputer. It is an actual computer, but it is so small that a trillion of them would fit in a test tube. It consists of DNA and DNA-processing enzymes, both dissolved in liquid; thus its input, output, and software are all in the form of DNA molecules. The purpose of the nanocomputer is to analyze DNA, detecting abnormalities in the human body and creating remedies for them.
Several early biotechnology firms were founded by scientists involved in fundamental research: Boyer, for example, teamed up with the venture capitalist Robert Swanson in 1976 to form Genentech (Genetic Engineering Technology). Other pioneering companies, including Cetus, Biogen, and Genex, likewise were founded through the collaboration of scientists and businesspeople. Today biotechnology promises a revolution in numerous areas, such as agriculture. Recombinant DNA techniques enable scientists to produce plants that are resistant to freezing temperatures, that will take longer to ripen, that will develop their own resistance to pests, and so on. By 1988 scientists had tested more than two dozen kinds of plants engineered to have special properties such as these. Yet no field of biotechnology and genetic engineering is as significant as the applications to health and the cures for diseases.
Medicines and Cures
The use of rDNA allows scientists to produce many products that were previously available only in limited quantities: for example, insulin, which we referred to earlier. Until the 1980s the only source of insulin for people with diabetes came from animals slaughtered for meat and other purposes. The supply was never high enough to meet demand, and this drove up prices. Then, in 1982, the U.S. Food and Drug Administration (FDA) approved the sale of insulin produced by genetically altered organisms—the first such product to become available. Since 1982 several additional products, such as human growth hormone, have been made with rDNA techniques.
One of the most exciting potential applications of genetic engineering is the treatment of genetic disorders, which are discussed in Heredity, through the use of gene therapy. Among the more than 3,000 such disorders, quite a few of which are quite serious or even fatal, many are the result of relatively minor errors in DNA sequencing. Genetic engineering offers the potential to provide individuals with correct copies of a gene, which could make possible a cure for that condition. In the 1980s scientists began clinical trials of a procedure known as human gene therapy to replace defective genes. The technique, still very much in the developmental stage, offers the hope of cures for diseases that medicine has long been powerless to combat.
In 2001 scientists at the Weizmann Institute in Israel brought together two of the most exciting fields of research, biotechnology and computers, to produce the DNA-processing nanocomputer. It is an actual computer, but it is so small that a trillion of them would fit in a test tube. It consists of DNA and DNA-processing enzymes, both dissolved in liquid; thus its input, output, and software are all in the form of DNA molecules. The purpose of the nanocomputer is to analyze DNA, detecting abnormalities in the human body and creating remedies for them.
Thursday, June 25, 2009
Principles of Genetic Engineering
Just as DNA is at the core of studies in genetics, recombinant DNA (rDNA)—that is, DNA that has been genetically altered through a process known as gene splicing—is the focal point of genetic engineering. In gene splicing, a DNA strand is cut in half lengthwise and joined with a strand from another organism or perhaps even another species. Use of gene splicing makes possible two other highly significant techniques. Gene transfer, or incorporation of new DNA into an organism's cells, usually is carried out with the help of a microorganism that serves as a vector, or carrier. Gene therapy is the introduction of normal or genetically altered genes to cells, generally to replace defective genes involved in genetic disorders.
DNA also can be cut into shorter fragments through the use of restriction enzymes. (An enzyme is a type of protein that speeds up chemical reactions.) The ends of these fragments have an affinity for complementary ends on other DNA fragments and will seek those out in the target DNA. By looking at the size of the fragment created by a restriction enzyme, investigators can determine whether the gene has the proper genetic code. This technique has been used to analyze genetic structures in fetal cells and to diagnose certain blood disorders, such as sickle cell anemia.
Gene Transfer
Suppose that a particular base-pair sequence carries the instruction "make insulin"; if a way could be found to insert that base sequence into the DNA of bacteria, for example, those bacteria would be capable of manufacturing insulin. This, in turn, would greatly improve the lives of people with type 1 diabetes, who depend on insulin shots to aid their bodies in processing blood sugar. (See Non-infectious Diseases for more about diabetes.)
Although the concept of gene transfer is relatively simple, its execution presents considerable technical obstacles. The first person to surmount these obstacles was the American biochemist Paul Berg (1926-), often referred to as the "father of genetic engineering." In 1973 Berg developed a method for joining the DNA from two different organisms, a monkey virus known as SV40 and a virus called lambda phage. Although the accomplishment was clearly a breakthrough, Berg's method was difficult. Then, later that year, the American biochemists Stanley Cohen (1922-) at Stanford University, and Herbert Boyer (1936-) at the University of California at San Francisco discovered an enzyme that greatly increased the efficiency of the Berg procedure. The gene-transfer technique developed by Berg, Boyer, and Cohen formed the basis for much of the ensuing progress in genetic engineering.
DNA also can be cut into shorter fragments through the use of restriction enzymes. (An enzyme is a type of protein that speeds up chemical reactions.) The ends of these fragments have an affinity for complementary ends on other DNA fragments and will seek those out in the target DNA. By looking at the size of the fragment created by a restriction enzyme, investigators can determine whether the gene has the proper genetic code. This technique has been used to analyze genetic structures in fetal cells and to diagnose certain blood disorders, such as sickle cell anemia.
Gene Transfer
Suppose that a particular base-pair sequence carries the instruction "make insulin"; if a way could be found to insert that base sequence into the DNA of bacteria, for example, those bacteria would be capable of manufacturing insulin. This, in turn, would greatly improve the lives of people with type 1 diabetes, who depend on insulin shots to aid their bodies in processing blood sugar. (See Non-infectious Diseases for more about diabetes.)
Although the concept of gene transfer is relatively simple, its execution presents considerable technical obstacles. The first person to surmount these obstacles was the American biochemist Paul Berg (1926-), often referred to as the "father of genetic engineering." In 1973 Berg developed a method for joining the DNA from two different organisms, a monkey virus known as SV40 and a virus called lambda phage. Although the accomplishment was clearly a breakthrough, Berg's method was difficult. Then, later that year, the American biochemists Stanley Cohen (1922-) at Stanford University, and Herbert Boyer (1936-) at the University of California at San Francisco discovered an enzyme that greatly increased the efficiency of the Berg procedure. The gene-transfer technique developed by Berg, Boyer, and Cohen formed the basis for much of the ensuing progress in genetic engineering.
Saturday, June 20, 2009
How does genetic engineering Works
Any discussion of genetics makes reference to DNA (deoxyribonucleic acid), a molecule that contains genetic codes for inheritance. DNA resides in chromosomes, threadlike structures found in the nucleus, or control center, of every cell in every living thing. Chromosomes themselves are made up of genes, which carry codes for the production of proteins. The latter, of which there are many thousands of different varieties, make up the majority of the human body's dry weight.
Although it is central to the latest advances in modern genetic research, DNA was discovered more than 130 years ago. In 1869 the Swiss biochemist Johann Friedrich Miescher (1844-1895) isolated a substance, containing both nitrogen and phosphorus, that separated into a protein and an acid molecule. He called it nucleic acid, and in this material he discovered DNA. Some 74 years would pass, however, before scientists recognized the function of the nucleic acid Miescher had discovered. Then, in 1944, a research team led by the Canadian-born American bacteriologist Oswald Avery (1877-1955) found that by taking DNA from one type of bacterium and inserting it into another, the second bacterium took on certain traits of the first. This experiment, along with other experiments and research, proved that DNA serves as a blueprint for the characteristics and functions of organisms.
The Double Helix
Nine years later, in 1953, the American biochemist James D. Watson (1928-) and the English biochemist Francis Crick (1916-) solved the mystery of DNA's structure and explained the means by which it provides necessary instructions at critical moments in the course of cell division and growth. They proposed a double helix, or spiral staircase, model, which linked the chemical bases of DNA in definite pairs. Using this twisted-ladder model, they were able to explain how the DNA molecule could duplicate itself, since each side of the ladder is identical to the other; if separated, each would serve as the template for the formation of its mirror image.
The sides of the DNA ladder are composed of alternating sugar and phosphate molecules, like links in a chain, and consist of four different chemical bases: adenine, guanine, cytosine, and thymine. The four letters designating these bases—A, G, C, and T—are the alphabet of the genetic code, and each rung of the DNA molecule is made up of a combination of two of these letters. Owing to specific chemical affinities, A always combines with T and C with G, to form what is called a base pair. Specific sequences of these base pairs, which are bonded to each other by atoms of hydrogen, constitute the genes.
Although it is central to the latest advances in modern genetic research, DNA was discovered more than 130 years ago. In 1869 the Swiss biochemist Johann Friedrich Miescher (1844-1895) isolated a substance, containing both nitrogen and phosphorus, that separated into a protein and an acid molecule. He called it nucleic acid, and in this material he discovered DNA. Some 74 years would pass, however, before scientists recognized the function of the nucleic acid Miescher had discovered. Then, in 1944, a research team led by the Canadian-born American bacteriologist Oswald Avery (1877-1955) found that by taking DNA from one type of bacterium and inserting it into another, the second bacterium took on certain traits of the first. This experiment, along with other experiments and research, proved that DNA serves as a blueprint for the characteristics and functions of organisms.
The Double Helix
Nine years later, in 1953, the American biochemist James D. Watson (1928-) and the English biochemist Francis Crick (1916-) solved the mystery of DNA's structure and explained the means by which it provides necessary instructions at critical moments in the course of cell division and growth. They proposed a double helix, or spiral staircase, model, which linked the chemical bases of DNA in definite pairs. Using this twisted-ladder model, they were able to explain how the DNA molecule could duplicate itself, since each side of the ladder is identical to the other; if separated, each would serve as the template for the formation of its mirror image.
The sides of the DNA ladder are composed of alternating sugar and phosphate molecules, like links in a chain, and consist of four different chemical bases: adenine, guanine, cytosine, and thymine. The four letters designating these bases—A, G, C, and T—are the alphabet of the genetic code, and each rung of the DNA molecule is made up of a combination of two of these letters. Owing to specific chemical affinities, A always combines with T and C with G, to form what is called a base pair. Specific sequences of these base pairs, which are bonded to each other by atoms of hydrogen, constitute the genes.
Thursday, June 18, 2009
genetic engineering
Artificial manipulation, modification, and recombination of DNA or other nucleic-acid molecules in order to modify an organism or population of organisms. The term initially meant any of a wide range of techniques for modifying or manipulating organisms through heredity and reproduction. Now the term denotes the narrower field of recombinant-DNA technology, or gene cloning, in which DNA molecules from two or more sources are combined, either within cells or in test tubes, and then inserted into host organisms in which they are able to reproduce. This technique is used to produce new genetic combination that are of value to science, medicine, agriculture, or industry. Through recombinant-DNA techniques, bacteria have been created that are capable of synthesizing human insulin, human interferon, human growth hormone, a hepatitis-B vaccine, and other medically useful substances. Recombinant-DNA techniques, combined with the development of a technique for producing antibodies in great quantity, have made an impact on medical diagnosis and cancer research. Plants have been genetically adjusted to perform nitrogen fixation and to produce their own pesticides. Bacteria capable of biodegrading oil have been produced for use in oil-spill cleanups. Genetic engineering also introduces the fear of adverse genetic manipulations and their consequences (e.g., antibiotic-resistant bacteria or new strains of disease)
Concept :
Genetic engineering is the alteration of genetic material by direct intervention in genetic processes with the purpose of producing new substances or improving functions of existing organisms. It is a very young, exciting, and controversial branch of the biological sciences. On the one hand, it offers the possibility of cures for diseases and countless material improvements to daily life. Hopes for the benefits of genetic engineering are symbolized by the Human Genome Project, a vast international effort to categorize all the genes in the human species. On the other hand, genetic engineering frightens many with its potential for misuse, either in Nazi-style schemes for population control or through simple bungling that might produce a biological holocaust caused by a man-made virus. Symbolic of the alarming possibilities is the furor inspired by a single concept on the cutting edge of genetic engineering: cloning.
Concept :
Genetic engineering is the alteration of genetic material by direct intervention in genetic processes with the purpose of producing new substances or improving functions of existing organisms. It is a very young, exciting, and controversial branch of the biological sciences. On the one hand, it offers the possibility of cures for diseases and countless material improvements to daily life. Hopes for the benefits of genetic engineering are symbolized by the Human Genome Project, a vast international effort to categorize all the genes in the human species. On the other hand, genetic engineering frightens many with its potential for misuse, either in Nazi-style schemes for population control or through simple bungling that might produce a biological holocaust caused by a man-made virus. Symbolic of the alarming possibilities is the furor inspired by a single concept on the cutting edge of genetic engineering: cloning.
Wednesday, June 17, 2009
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If you require any more information or have any questions about our privacy policy, please feel free to contact us by email at hardrocker_9990@yahoo.co.in.
At www.cellsandgenetics.blogspot.com, the privacy of our visitors is of extreme importance to us. This privacy policy document outlines the types of personal information is received and collected by www.cellsandgenetics.blogspot.com and how it is used.
Log Files
Like many other Web sites, www.cellsandgenetics.blogspot.com makes use of log files. The information inside the log files includes internet protocol ( IP ) addresses, type of browser, Internet Service Provider ( ISP ), date/time stamp, referring/exit pages, and number of clicks to analyze trends, administer the site, track user’s movement around the site, and gather demographic information. IP addresses, and other such information are not linked to any information that is personally identifiable.
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