essay questions on recombinant dna technology

The pharmaceutical industry is another frontier for the use of GMOs. In 1986, human growth hormone was the first protein pharmaceutical made in plants (Barta et al ., 1986), and in 1989, the first antibody was produced (Hiatt et al ., 1989). Both research groups used tobacco, which has since dominated the industry as the most intensively studied and utilized plant species for the expression of foreign genes (Ma et al ., 2003). As of 2003, several types of antibodies produced in plants had made it to clinical trials. The use of genetically modified animals has also been indispensible in medical research. Transgenic animals are routinely bred to carry human genes, or mutations in specific genes, thus allowing the study of the progression and genetic determinants of various diseases.

Potential GMO Applications

Many industries stand to benefit from additional GMO research. For instance, a number of microorganisms are being considered as future clean fuel producers and biodegraders. In addition, genetically modified plants may someday be used to produce recombinant vaccines. In fact, the concept of an oral vaccine expressed in plants (fruits and vegetables) for direct consumption by individuals is being examined as a possible solution to the spread of disease in underdeveloped countries, one that would greatly reduce the costs associated with conducting large-scale vaccination campaigns. Work is currently underway to develop plant-derived vaccine candidates in potatoes and lettuce for hepatitis B virus (HBV), enterotoxigenic Escherichia coli (ETEC), and Norwalk virus. Scientists are also looking into the production of other commercially valuable proteins in plants, such as spider silk protein and polymers that are used in surgery or tissue replacement (Ma et al ., 2003). Genetically modified animals have even been used to grow transplant tissues and human transplant organs, a concept called xenotransplantation. The rich variety of uses for GMOs provides a number of valuable benefits to humans, but many people also worry about potential risks.

Risks and Controversies Surrounding the Use of GMOs

Despite the fact that the genes being transferred occur naturally in other species, there are unknown consequences to altering the natural state of an organism through foreign gene expression . After all, such alterations can change the organism's metabolism , growth rate, and/or response to external environmental factors. These consequences influence not only the GMO itself, but also the natural environment in which that organism is allowed to proliferate. Potential health risks to humans include the possibility of exposure to new allergens in genetically modified foods, as well as the transfer of antibiotic-resistant genes to gut flora.

Horizontal gene transfer of pesticide, herbicide, or antibiotic resistance to other organisms would not only put humans at risk , but it would also cause ecological imbalances, allowing previously innocuous plants to grow uncontrolled, thus promoting the spread of disease among both plants and animals. Although the possibility of horizontal gene transfer between GMOs and other organisms cannot be denied, in reality, this risk is considered to be quite low. Horizontal gene transfer occurs naturally at a very low rate and, in most cases, cannot be simulated in an optimized laboratory environment without active modification of the target genome to increase susceptibility (Ma et al ., 2003).

In contrast, the alarming consequences of vertical gene transfer between GMOs and their wild-type counterparts have been highlighted by studying transgenic fish released into wild populations of the same species (Muir & Howard, 1999). The enhanced mating advantages of the genetically modified fish led to a reduction in the viability of their offspring . Thus, when a new transgene is introduced into a wild fish population, it propagates and may eventually threaten the viability of both the wild-type and the genetically modified organisms.

Unintended Impacts on Other Species: The Bt Corn Controversy

One example of public debate over the use of a genetically modified plant involves the case of Bt corn. Bt corn expresses a protein from the bacterium Bacillus thuringiensis . Prior to construction of the recombinant corn, the protein had long been known to be toxic to a number of pestiferous insects, including the monarch caterpillar, and it had been successfully used as an environmentally friendly insecticide for several years. The benefit of the expression of this protein by corn plants is a reduction in the amount of insecticide that farmers must apply to their crops. Unfortunately, seeds containing genes for recombinant proteins can cause unintentional spread of recombinant genes or exposure of non-target organisms to new toxic compounds in the environment.

The now-famous Bt corn controversy started with a laboratory study by Losey et al . (1999) in which the mortality of monarch larvae was reportedly higher when fed with milkweed (their natural food supply) covered in pollen from transgenic corn than when fed milkweed covered with pollen from regular corn. The report by Losey et al . was followed by another publication (Jesse & Obrycki, 2000) suggesting that natural levels of Bt corn pollen in the field were harmful to monarchs.

Debate ensued when scientists from other laboratories disputed the study, citing the extremely high concentration of pollen used in the laboratory study as unrealistic, and concluding that migratory patterns of monarchs do not place them in the vicinity of corn during the time it sheds pollen. For the next two years, six teams of researchers from government, academia, and industry investigated the issue and concluded that the risk of Bt corn to monarchs was "very low" (Sears et al ., 2001), providing the basis for the U.S. Environmental Protection Agency to approve Bt corn for an additional seven years.

Unintended Economic Consequences

Another concern associated with GMOs is that private companies will claim ownership of the organisms they create and not share them at a reasonable cost with the public. If these claims are correct, it is argued that use of genetically modified crops will hurt the economy and environment, because monoculture practices by large-scale farm production centers (who can afford the costly seeds) will dominate over the diversity contributed by small farmers who can't afford the technology. However, a recent meta-analysis of 15 studies reveals that, on average, two-thirds of the benefits of first-generation genetically modified crops are shared downstream, whereas only one-third accrues upstream (Demont et al ., 2007). These benefit shares are exhibited in both industrial and developing countries. Therefore, the argument that private companies will not share ownership of GMOs is not supported by evidence from first-generation genetically modified crops.

GMOs and the General Public: Philosophical and Religious Concerns

In a 2007 survey of 1,000 American adults conducted by the International Food Information Council (IFIC), 33% of respondents believed that biotech food products would benefit them or their families, but 23% of respondents did not know biotech foods had already reached the market. In addition, only 5% of those polled said they would take action by altering their purchasing habits as a result of concerns associated with using biotech products.

According to the Food and Agriculture Organization of the United Nations, public acceptance trends in Europe and Asia are mixed depending on the country and current mood at the time of the survey (Hoban, 2004). Attitudes toward cloning, biotechnology, and genetically modified products differ depending upon people's level of education and interpretations of what each of these terms mean. Support varies for different types of biotechnology; however, it is consistently lower when animals are mentioned.

Furthermore, even if the technologies are shared fairly, there are people who would still resist consumable GMOs, even with thorough testing for safety, because of personal or religious beliefs. The ethical issues surrounding GMOs include debate over our right to "play God," as well as the introduction of foreign material into foods that are abstained from for religious reasons. Some people believe that tampering with nature is intrinsically wrong, and others maintain that inserting plant genes in animals, or vice versa, is immoral. When it comes to genetically modified foods, those who feel strongly that the development of GMOs is against nature or religion have called for clear labeling rules so they can make informed selections when choosing which items to purchase. Respect for consumer choice and assumed risk is as important as having safeguards to prevent mixing of genetically modified products with non-genetically modified foods. In order to determine the requirements for such safeguards, there must be a definitive assessment of what constitutes a GMO and universal agreement on how products should be labeled.

These issues are increasingly important to consider as the number of GMOs continues to increase due to improved laboratory techniques and tools for sequencing whole genomes, better processes for cloning and transferring genes, and improved understanding of gene expression systems. Thus, legislative practices that regulate this research have to keep pace. Prior to permitting commercial use of GMOs, governments perform risk assessments to determine the possible consequences of their use, but difficulties in estimating the impact of commercial GMO use makes regulation of these organisms a challenge.

History of International Regulations for GMO Research and Development

In 1971, the first debate over the risks to humans of exposure to GMOs began when a common intestinal microorganism, E. coli , was infected with DNA from a tumor-inducing virus (Devos et al ., 2007). Initially, safety issues were a concern to individuals working in laboratories with GMOs, as well as nearby residents. However, later debate arose over concerns that recombinant organisms might be used as weapons. The growing debate, initially restricted to scientists, eventually spread to the public, and in 1974, the National Institutes of Health (NIH) established the Recombinant DNA Advisory Committee to begin to address some of these issues.

In the 1980s, when deliberate releases of GMOs to the environment were beginning to occur, the U.S. had very few regulations in place. Adherence to the guidelines provided by the NIH was voluntary for industry. Also during the 1980s, the use of transgenic plants was becoming a valuable endeavor for production of new pharmaceuticals, and individual companies, institutions, and whole countries were beginning to view biotechnology as a lucrative means of making money (Devos et al ., 2007). Worldwide commercialization of biotech products sparked new debate over the patentability of living organisms, the adverse effects of exposure to recombinant proteins, confidentiality issues, the morality and credibility of scientists, the role of government in regulating science, and other issues. In the U.S., the Congressional Office of Technology Assessment initiatives were developed, and they were eventually adopted worldwide as a top-down approach to advising policymakers by forecasting the societal impacts of GMOs.

Then, in 1986, a publication by the Organization for Economic Cooperation and Development (OECD), called "Recombinant DNA Safety Considerations," became the first intergovernmental document to address issues surrounding the use of GMOs. This document recommended that risk assessments be performed on a case-by-case basis. Since then, the case-by-case approach to risk assessment for genetically modified products has been widely accepted; however, the U.S. has generally taken a product-based approach to assessment, whereas the European approach is more process based (Devos et al ., 2007). Although in the past, thorough regulation was lacking in many countries, governments worldwide are now meeting the demands of the public and implementing stricter testing and labeling requirements for genetically modified crops.

Increased Research and Improved Safety Go Hand in Hand

Proponents of the use of GMOs believe that, with adequate research, these organisms can be safely commercialized. There are many experimental variations for expression and control of engineered genes that can be applied to minimize potential risks. Some of these practices are already necessary as a result of new legislation, such as avoiding superfluous DNA transfer (vector sequences) and replacing selectable marker genes commonly used in the lab (antibiotic resistance) with innocuous plant-derived markers (Ma et al ., 2003). Issues such as the risk of vaccine-expressing plants being mixed in with normal foodstuffs might be overcome by having built-in identification factors, such as pigmentation, that facilitate monitoring and separation of genetically modified products from non-GMOs. Other built-in control techniques include having inducible promoters (e.g., induced by stress, chemicals, etc.), geographic isolation, using male-sterile plants, and separate growing seasons.

GMOs benefit mankind when used for purposes such as increasing the availability and quality of food and medical care, and contributing to a cleaner environment. If used wisely, they could result in an improved economy without doing more harm than good, and they could also make the most of their potential to alleviate hunger and disease worldwide. However, the full potential of GMOs cannot be realized without due diligence and thorough attention to the risks associated with each new GMO on a case-by-case basis.

References and Recommended Reading

Barta, A., et al . The expression of a nopaline synthase-human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Molecular Biology 6 , 347–357 (1986)

Beyer, P., et al . Golden rice: Introducing the β-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency. Journal of Nutrition 132 , 506S–510S (2002)

Demont, M., et al . GM crops in Europe: How much value and for whom? EuroChoices 6 , 46–53 (2007)

Devlin, R., et al . Extraordinary salmon growth. Nature 371 , 209–210 (1994) ( link to article )

Devos, Y., et al . Ethics in the societal debate on genetically modified organisms: A (re)quest for sense and sensibility. Journal of Agricultural and Environmental Ethics 21 , 29–61 (2007) doi:10.1007/s10806-007-9057-6

Guerrero-Andrade, O., et al . Expression of the Newcastle disease virus fusion protein in transgenic maize and immunological studies. Transgenic Research 15 , 455–463(2006) doi:10.1007/s11248-006-0017-0

Hiatt, A., et al . Production of antibodies in transgenic plants. Nature 342 , 76–79 (1989) ( link to article )

Hoban, T. Public attitudes towards agricultural biotechnology. ESA working papers nos. 4-9. Agricultural and Development Economics Division, Food and Agricultural Organization of the United Nations (2004)

Jesse, H., & Obrycki, J. Field deposition of Bt transgenic corn pollen: Lethal effects on the monarch butterfly. Oecologia 125 , 241–248 (2000)

Losey, J., et al . Transgenic pollen harms monarch larvae. Nature 399 , 214 (1999) doi:10.1038/20338 ( link to article )

Ma, J., et al . The production of recombinant pharmaceutical proteins in plants. Nature Reviews Genetics 4 , 794–805 (2003) doi:10.1038/nrg1177 ( link to article )

Muir, W., & Howard, R. Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan gene hypothesis. Proceedings of the National Academy of Sciences 96 , 13853–13856 (1999)

Sears, M., et al . Impact of Bt corn on monarch butterfly populations: A risk assessment. Proceedings of the National Academy of Sciences 98 , 11937–11942 (2001)

Spurgeon, D. Call for tighter controls on transgenic foods. Nature 409 , 749 (2001) ( link to article )

Takeda, S., & Matsuoka, M. Genetic approaches to crop improvement: Responding to environmental and population changes. Nature Reviews Genetics 9 , 444–457 (2008) doi:10.1038/nrg2342 ( link to article )

United States Department of Energy, Office of Biological and Environmental Research, Human Genome Program. Human Genome Project information: Genetically modified foods and organisms, (2007)

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Recombinant DNA Technology ( AQA A Level Biology )

Revision note.

Biology Lead

Recombinant DNA Technology

Recombinant dna.

• The genetic code is universal , meaning that almost every organism uses the same four nitrogenous bases – A, T, C & G. There are a few exceptions
• The genetic code is the basis for storing instructions that, alongside environmental influences, dictate the behaviour of cells and as a result, the behaviour of the whole organism
• The universal nature of the genetic code means that the same codons code for the same amino acids in all living things (meaning that genetic information is transferable between species)
• Thus scientists have been able to artificially change an organism's DNA by combining lengths of nucleotides from different sources (typically the nucleotides are from different species)
• The altered DNA, with the introduced nucleotides, is called recombinant DNA (rDNA)
• If an organism contains nucleotide sequences from a different species it is called a transgenic organism
• Any organism that has introduced genetic material is a genetically modified organism (GMO)
• The mechanisms of transcription and translation are also universal which means that the transferred DNA can be translated within cells of the genetically modified organism

Illustration of a maize plant that has recombinant DNA (DNA from Bacillus thuringiensis) .

Recombinant DNA technology

• This form of genetic engineering involves the transfer of fragments of DNA from one organism/species into another organism/species
• The resulting genetically engineered organism will then contain recombinant DNA and will be a genetically modified organism (GMO)
• Isolation of the desired DNA fragment
• Multiplication of the DNA fragment (using polymerase chain reaction - PCR)
• Transfer into the organism using a vector (e.g. plasmids, viruses, liposomes)
• Identification of the cells with the new DNA fragment (by using a marker ), which is then cloned
• Enzymes (restriction endonucleases, ligase and reverse transcriptase)
• Vectors - used to deliver DNA fragments into a cell (eg. plasmids, viruses and liposomes)
• Markers - genes that code for identifiable substances that can be tracked (eg. GFP - green fluorescent protein which fluoresces under UV light or GUS - β-glucuronidase enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent)
• This is an area of research that studies the design and construction of different biological pathways, organisms and devices, as well as the redesigning of existing natural biological systems

An overview of the steps taken to genetically engineer an organism (in this case bacteria are being genetically engineered to produce human insulin)

When answering questions about genetic engineering you should remember to include the names of any enzymes ( restriction endonucleases , reverse transcriptase , ligase ) involved and mention that markers (genes which can be identified) and vectors (transfer the desired gene) are also used.

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Techniques of Recombinant DNA Technology | Essay | Biotechnology

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Techniques of Recombinant DNA Technology

Essay Contents:

• Essay on Down Streaming Processing

ADVERTISEMENTS: (adsbygoogle = window.adsbygoogle || []).push({}); Essay # 1. Isolation of Genetic Material:

Following steps are required for isolation of genetic material DNA in pure form:

(i) Bacterial cells or plant cells or animal cells are treated with enzymes like lysozyme for bacteria cellulose for plant cells and chitinase for fungus. This will break the cell envelope open and like DNA, RNA, proteins, polysaccharides and lipids will be released.

(ii) As in eukaryotic cells DNA is interwined with protein molecules like histones. Additional Protein can be removed by treating with enzyme protease. RNA can be removed by treating with enzyme ribonuclease.

(iii) DNA sample can be further purified by using additional extraction techniques.

(iv) By addition of chilled ethanol, DNA Precipitates out and can be observed in the form of fine threads m suspension.

Agarose gel electrophoresis is employed to check the progression of restriction enzyme digestion. DNA being negatively charged moves towards anode (positive electrode). Same technique is used for vector DNA.

Essay # 2. Amplification of Gene of Interest Using PCR:

In PCR (Polymerase Chain Reaction) multiple copies of desired DNA (gene) can be formed in vitro.

Polymerase Chain Reaction (PCR) (Specific Sequences can be Amplified):

PCR technique was developed by Kary Mullis in 1985. If one knows the sequence of at least part of a DNA segment to be cloned, a number of copies of that DNA Sent can be hugely amplified using polymerase chain reaction. It is able to generate microgram (µg) quantities of DNA copies (up to billion copies) of desired DNA segment, present even as a single copy with in short time.

The technique is based on principle that when a DNA molecule is subjected to high temperature due to denaturation the two DNA strands separate. As a result two single stranded DNA molecules appear. DNA polymerase can copy these single stranded molecules. This leads to the formation of original DNA double stranded molecule. Due to repetition of this process several copies of DNA sequences can be formed.

Steps involved in PCR reaction:

Basic requirements for PCR reaction are:

(i) DNA template (desired segment) to be amplified,

(ii) Two nucleotide primers (usually 10-18 nucleotides long) specific i.e., complementary to the sequences present at the 3 ends of the desired DNA segment. These primers are oriented with their ends facing each other permitting formation of DNA between them.

(iii) High temperature (790°C) stable DNA polymerase. It is needed for the formation of new DNA Usually used DNA polymerase for PCR reactions is Taq polymerase.

a. Isolated target DNA segment to be amplified is heated to high temperature (94°C) for denaturation. It leads to separation of two DNA strands.

b. Next step is of annealing. Here each single strand of target DNA acts as template for DNA synthesis. It is cooled (40°-60C) in presence of large excess of synthetic oligonucleotide primers. In annealing two oligonucleotide primers anneal or hybridize to each of single stranded template DNA. Annealing sequences are located at 3′ end of two strands of desired segment.

c. It is followed by extension step. Here Taq DNA polymerase synthesizes the complementary strand by using, 3’-OH of primer. The primers extend towards each other so that DNA segment lying between two primers is copied. This step requires dNTPs (deoxynucleoside triphosphates) and Mg 2+ . Temperature required for this step is 72°C.

The enzyme extends the primers using the genomic DNA as template and nucleotides made available in reaction. Here DNA replication occurs several times. Segment DNA gets amplified to approximately billion times.

As a result 9 billion copies can be formed. Repeated amplification is possible by use of thermostable DNA polymerase. It is isolated from bacterium, Thermus aquaticus. This enzyme remains active during high temperature induced denaturation of double helix DNA.

Essay # 3. Preparation of the Gene:

Gene coining in bacteria is achieved by cleaving the purified DNA with enzyme restriction endonuclease which produces small fragments (approximately 4 kilobase pairs). Each fragment has a stickly’ complementary single-stranded end. Eukaryotic genes contain introns that are not processed in bacteria therefore; DNA for cloning is usually obtained as a reverse transcriptase generated copy DNA (cDNA) of the relevant mRNA. In cases where nucleotide or amino acid sequences are known, synthetic DNA may be produced.

The vector is an agent which is used to transfer DNA into a host cell e.g., plasmid, bacteriophage. The vector is cut with the same enzyme (restriction endonuclease) as that used to generate the chromosomal DNA fragments. The chromosomal fragments and linearised vector are incubated with DNA ligase which covalently joins the DNA molecules (Fig. 11.21). Those plasmids which contain an inserted fragment are called recombinant plasmid.

Transformation of Host Cell:

The ligated plasmid mixture is introduced into the bacterial cell where they take up DNA through transformation process. Transformation is generally carried out by placing actively growing cells of a bacterium in cold, dilute solution of CaCl 2 which enhances the ability of bacterial cells to take up foreign DNA.

In majority of the cases, E. coli is the most preferred host because:

(i) Molecular biology of this bacterium is well understood,

(ii) Calcium chloride treated cells are highly transformable, and

(iii) E. coli transcribes and translates most Gram-positive and Gram-negative genes except some actinomycete genes.

There are many methods to introduce the ligated DNA into recipient competent cell. Suppose a recombinant DNA having ampicillin antibiotic resistant gene is transferred to E. coli cells, the host cells become transformed into ampicillin resistant cells. When such cells are spread over agar plates containing ampicillin on transformants will grow there. Ampicillin resistance gene is called ‘selectable marker’ as due to this transformed cell can be selected in presence of ampicillin.

Essay # 4. Detection of the Cloned Gene (Recombinants):

Cells with recombinant DNA (rDNA) are selected on the expression or non-expression of some traits like resistance to antibiotic chloramphenicol. Direct selection of recombinants is made due to encoding of these traits by vector or cloned DNA sequence.

Various methods for identification of recombinants are:

1. Transformants (host cells with foreign DNA) can be selected by:

(i) Host cells transformed with plasmid having ampicillin resistant gene are grown on medium having antibiotic ampicillin, only those cells bearing the above plasmid will be able to grow on it.

(ii) But one is not able to know that which colony bear recombinant plasmid and which bear relegated vector plasmid.

2. Insertional Inactivation Method:

It is based on basic principle that cloned DNA fragment disrupts the coding sequence of gene.

To identify recombinants, one of the important approaches is to use DNA probe. In a DNA molecule, the two complementary strands are held together by hydrogen bonds. If two similar DNA pieces are mixed together and hydrogen bonds broken (by heating) the strands will separate.

Upon lowering the temperature, the hydrogen bonds are formed again. Some of the resultant double-stranded DNA will be hybrids i.e., composed of one strand of one type and one strand of the other type. This concept of DNA hybridization has been exploited for utilizing the DNA molecules as probes (Fig. 11.22).

The transformed colonies are replica plated to a nitrocellulose filter and are lysed to release the DNA. This DNA is denatured (by raising the temperature) and fixed to the nitrocellulose so as to produce a DNA print corresponding exactly to the position of the colonies on the original plate.

The DNA print is then hybridized with the probe which has been previously radioactively labelled. After washing off unhybridized DNA, the position of the radioactive spots on the filter is indicated by autoradiography in order to identify the presence of the required DNA.

Essay # 5 . Obtaining the Foreign Gene Product:

When alien DNA is inserted into cloning vector and then transferred to host cell, DNA gets multiplied. Due to expression of this foreign gene, proteins can be formed.

Such target proteins (recombinant proteins) are to be produced in large scale. The cells having cloned genes may be grown on small scale in laboratory. Such cultures are used to make proteins with required characters. For multiplication of cells continuous culture for system is used. Here medium is drained out and fresh medium is added. This helps in active growth of cells during log/exponential phase.

To produce this product in large quantities bioreactors are needed. In such bioreactors 100-1000 litres of culture can be processed.

Most commonly used in stirring type bioreactor, whose details are given below?

Fermenter (Bioreactor):

The basic design of a stirred-tank fermenter is shown in Fig. 11.23. It consists of a large stainless steel vessel with a capacity of upto 500,000 dm 3 around which there is a jacket of circulatory water used to control the temperature within the fermenter. There is also an agitator, comprising of a series of flat blades, which can be rotated with the help of a motor. This ensures the thoroughly mixing of the contents so that nutrients come in close with the micro-organisms. The agitator also prevents settling out of the cells at the bottom.

Fermenter also has adequate arrangement for aeration, temperature and pH control. For proper aeration, air can be forced in at the bottom of the tank through a porous ring, called sparger, by the process called sparging, while there is an outlet to remove air and waste gases at the top of the tank.

The top of the tank also a number of inlet tubes called ports, through which materials can be introduced or withdrawn e.g:

i. Inoculation port for introducing initial inoculum;

ii. Nutrient port for introducing more nutrients;

iii. Antifoam port for introducing antifoaming agents; and

iv. pH port for introducing acid or alkali to maintain optimal pH.

At the base of the tank, there is a harvest line to extract culture medium and microbial products. To regularly detect the pH and temperature changes, tank is fitted with certain probes.

Significance:

The stirred-tank fermenter is a well- tried and tested design for large-scale production of micro-organisms under aseptic and controlled environment for a number of days. Small-scale fermenters of 10-100 litres capacity are used in research laboratories. It is also provided with many controls for the monitoring of physical, chemical and biological parameters that affect the growth of cells.

It is relatively costly to run largely due to high energy requirements to drive the agitators and introduce the compressed air.

Essay # 6. Down Streaming Processing:

Products formed are separated and purified. Steps are collectively called as down streaming processing. Suitable preservatives are used. For medicinal purposes, clinical trials are carried out. Quality control is also maintained.

Related Articles:

• 7 Main Stages of Recombinant DNA Technology
• Recombinant DNA Technology (With Diagram)

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Article Contents

Acknowledgements.

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Personal Reflections on the Origins and Emergence of Recombinant DNA Technology

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Paul Berg, Janet E Mertz, Personal Reflections on the Origins and Emergence of Recombinant DNA Technology, Genetics , Volume 184, Issue 1, 1 January 2010, Pages 9–17, https://doi.org/10.1534/genetics.109.112144

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The emergence of recombinant DNA technology occurred via the appropriation of known tools and procedures in novel ways that had broad applications for analyzing and modifying gene structure and organization of complex genomes. Although revolutionary in their impact, the tools and procedures per se were not revolutionary. Rather, the novel ways in which they were applied was what transformed biology.

Anecdotal, Historical and Critical Commentaries on Genetics

FREEMAN Dyson contrasts what he called the “Kuhnian” and “Galisonian” views of the origins of scientific revolutions in a review of Peter Galison's book, Einstein's Clocks, Poincare's Maps: Empires of Time ( Dyson 2003 ). In The Structure of Scientific Revolutions , Thomas Kuhn proposes that revolutionary breakthroughs in science are triggered primarily by ideas that, by their novelty, transform or replace the prevailing paradigm ( Kuhn 1962 ). By contrast, Galison (2003) attributes such breakthroughs to new tools that, by their nature, make possible new approaches to formerly intractable problems. Galison also acknowledges that the application of existing tools in novel ways often provides the means to explore what was previously impossible. As Alfred Hershey has been quoted saying, “There is nothing more satisfying to me than developing a method. Ideas come and go, but a method lasts” ( Stahl 1998 ).

The Galison view is exemplified by the genetic revolution in biotechnology, which relied on both the discovery of new tools and the use of existing tools in new ways. The key methodological advances were: (i) the discovery of enzymes that modify DNA molecules in ways that enable them to be joined together in new combinations; (ii) the demonstration that DNA molecules can be cloned, propagated, and expressed in bacteria; (iii) the development of methods for chemically synthesizing and sequencing DNA molecules; and (iv) the development of the polymerase chain reaction method for amplifying DNA in vitro .

Although the emergence of recombinant DNA technology was transformational in its impact, the tools and procedures that were the keys to its development largely emerged as enhancements and extensions of existing knowledge, i.e. , they were evolutionary, not revolutionary, in nature. What was novel was the numerous ways in which many investigators applied these technologies for analyzing and modifying gene structure and the organization of complex genomes. Especially striking was the rapidity with which the new technologies took hold and dominated research into many different biological problems. Today, recombinant DNA technology has altered the ways both questions are formulated and solutions are sought. Scientists now routinely isolate genes from any organism on our planet, alive or dead. The construction of new variants of genes, chromosomes, and viruses has become standard practice in research laboratories. Only science fiction one-half century ago, the introduction of new genes into microbes, plants, and animals, including humans, is a common occurrence. The tools of recombinant DNA greatly expedite sequencing of the genomes of humans and numerous other species. Along with these advances have come astonishing improvements in medical diagnoses, prognoses, and therapies. In addition, many commercial opportunities have been realized, with the United States being the world leader in the biotechnology industry. Equally profound is the influence these developments have had on many related fields. Even a cursory look at journals in such diverse fields as chemistry, evolutionary biology, paleontology, anthropology, linguistics, psychology, medicine, plant science, and, even, forensics, information theory, and computer science shows the pervasive influence of this new technology. This essay traces the conceptual and experimental origins of the recombinant DNA technology.

Background:

During the 1960s, enormous progress was made in understanding the structure of genes and the mechanisms of their replication, expression, and regulation in prokaryotes and the viruses that infect them. However, largely unknown at the end of that decade was whether these findings were applicable to eukaryotes, i.e ., organisms with an authentic nucleus, and, in particular, mammalian cells. The reason was that the experimental tools available at that time for exploring the molecular and genetic properties of mammalian organisms were woefully inadequate for the task.

One method that had been very powerful in investigations of the molecular biology of the most widely studied microbe, Escherichia coli , was the property of bacteriophage (commonly abbreviated “phage”) to transfer genes from one strain to another, a process referred to as transduction. For example, in the case of “generalized transduction” by phage P1, E. coli cells are infected with phage P1, the viral proteins are synthesized, the viral genome is replicated, and new infectious virus particles are assembled. However, concomitant with virus multiplication, random segments of the infected cell's DNA are also incorporated into newly formed virus particles in place of viral DNA. When such a pseudo-P1 phage “infects” a bacterium, neither virus replication nor cell death occurs. Instead, the bacterial DNA contained within the pseudo-P1 phage enters the bacterium and recombines at low frequency with the cell's chromosome to become a permanent part of that cell's genetic makeup. If the newly acquired bacterial DNA confers a measurable or selectable property, the rare recombinant can be recovered using an appropriate selection condition. In this way, any part of the genome of one E. coli strain can be transferred to the genome of another E. coli strain. Zinder's recollections of his and Lederberg's discovery of bacteriophage-mediated gene transfer in bacteria has been described in an earlier Perspectives article ( Zinder 1992 ).

An alternate way of transferring genes from one E. coli cell to another is exemplified by phage λ-mediated “specialized transduction.” In this system, transduction occurs when the phage DNA integrates into the infected cell's chromosome, and bacterial DNA adjacent to the site of integration is excised and packaged into phage particles along with the viral DNA. The cellular DNA acquired by the phage can then be transferred to new hosts during subsequent rounds of infection. These two modes of virus-mediated transduction are distinctive in that phage P1 can transfer DNA from any region of the bacterial chromosome while phage λ transfers only regions of the bacterial chromosome adjacent to sequence-specific phage λ integration sites ( Campbell 2007 ).

It seemed reasonable to consider whether a comparable virus-mediated gene-transfer system exists for mammalian cells. The small DNA viruses, polyoma and SV40, were deemed to be good candidates. It was already known that infection of cultured mouse cells with polyoma virus results in the production of infectious polyoma progeny and virus particles containing exclusively mouse DNA. Importantly, the mouse DNA contained in these polyoma “pseudovirions” is representative of the entire mouse genome. A similar finding was made with the related primate virus, SV40. However, in this case, some virus particles are produced in which host cellular DNA is covalently joined to the viral DNA. Might it be possible, we mused, that polyoma or SV40 could be used to transfer genes from one mammalian cell to another in much the same way that phage transfer genes among bacteria? On the face of it, that seemed unlikely for the following reasons. The amount of bacterial DNA that can be accommodated in a phage P1 particle is ∼2% of the E. coli genome; somewhat less cellular DNA can be transferred by phage λ. By contrast, polyoma and SV40 virions can accommodate only 5–6 kbp of DNA, i.e. , roughly one-millionth of a mammalian genome. Thus, the probability of acquiring a specific mammalian gene in a polyoma or SV40 virion particle is at least four orders of magnitude lower than is the probability that a P1 or λ phage particle will contain one or more specific E. coli genes. In addition, the difficulty of picking out a specific, unique segment of mammalian DNA without having on hand a very strong method of selection or detection made the whole notion rather infeasible.

An alternative that seemed worth exploring was whether specific segments of mammalian, or any DNA for that matter, could be recombined with SV40 DNA in vitro . That would bypass the need for the recombinant product to be incorporated into a virus particle. This idea was attractive because mammalian cells have the capacity to take up “naked” DNA such as the SV40 genome, integrating it into the host cell's genome. Thus, any DNA covalently linked to SV40 DNA could become integrated into the chromosomes of a mammalian cell along with the viral DNA. In theory, such cells could be screened or selected for the presence and expression of both the SV40 and foreign DNAs. Thus, the first step toward achieving this game plan involved devising a method for introducing foreign DNA into the SV40 genome.

In early 1971, the American Cancer Society approved a grant application in which Berg proposed to develop the means for transducing foreign DNA into mammalian cells ( Berg 1970 ). In the proposal, he identified SV40 DNA as the vector because it can be taken up by rodent and primate cells, including human ones, where it can replicate to high copy number as an autonomous plasmid or integrate into the host cell's genome. For the recombinant partner, the DNA would, ideally, be one (i) whose integration and possible expression in mammalian cells could be assayed, (ii) that could replicate as an autonomous plasmid in E. coli , and (iii) that has a gene whose expression could provide a way to screen or select E. coli cells containing the DNA.

But first, a method for joining together two DNAs in vitro needed to be developed. The plan was based on the knowledge that the bacteriophage λ genome exists as a linear DNA molecule within its virus particle, yet becomes a circular molecule following infection of its host, E. coli . That property stems from the existence of complementary, single-stranded extensions on the 5′-ends of the linear phage λ DNA enabling the ends to be joined ( Hershey et al . 1963 ). At low DNA concentration, intramolecular base pairing of these complementary single-stranded ends leads primarily to the formation of monomeric circular DNA molecules; at high DNA concentration, intermolecular end-to-end joining leads primarily to the formation of oligomeric DNA molecules. Such complementary ends are referred to as being “cohesive” or “sticky.” Furthermore, these hydrogen-bonded rings can be sealed in vitro by incubation with DNA ligase to create covalently closed circular DNA molecules ( Gellert et al . 1968 ; Wu and Kaiser 1968 ). Thus, it seemed attractive to consider constructing “artificial” cohesive ends as the strategy for joining together two different DNAs.

Following that strategy required a procedure for constructing short stretches of complementary nucleotides onto the ends of the two molecules to be recombined and to rely on their capacity to base pair in vitro to effect the joining. The enzyme terminal deoxynucleotidyl transferase (TdT) seemed admirably suited for this purpose since it was known to synthesize chains of a single nucleotide onto the 3′-ends of duplex DNA when a single nucleoside triphosphate is provided as the nucleotidyl donor ( Kato et al. 1967 ). Synthesizing short polynucleotide chains of adenylates onto the 3′-ends of one DNA and approximately the same length polynucleotide chains of thymidylates onto the 3′-ends of the other DNA would create the necessary cohesive ends for joining together two DNA molecules. David Jackson 4 and Robert Symons 5 undertook the task of exploring this approach.

Peter Lobban 6 independently conceived the idea of using a series of enzymes to covalently join DNAs together in vitro while fulfilling the Stanford Biochemistry Department's requirement for Ph.D. students to write and defend an original research proposal ( Lobban 1969 ).

Lobban's stated goal was to create a λ phage-based transduction system by replacing nonessential DNA in the middle of the phage λ genome with “foreign” DNA ( Figure 1 ). He proposed to isolate DNA segments derived from the left and right “arms” of λ phage DNA and then to join the foreign DNA to the two internal ends of these arms. The cohesive ends present on the left and right arms would be left intact to permit the recombinant genome to circularize and replicate. The formation of the recombinant was to be achieved by using TdT to add short polymeric tails to the 3′-ends of the foreign DNA and complementary polymeric tails to the internal 3′-ends of the left and right arms of the λ DNA.

“Steps in the creation of transducing genomes (digestion with λ exonuclease not shown),” the procedure originally proposed by Lobban for inserting “foreign DNA” into the left and right arms of phage λ DNA in vitro . Reproduced from Figure 3 of Lobban (1969) .

In his proposal and Ph.D. thesis ( Lobban 1972 , Lobban ) foresaw the prospect of inserting any foreign DNA, including from mammalian cells, into the phage DNA. He suggested that such an approach might enable specific mammalian genes to be identified and their mRNA and protein products to be detected and recovered in E. coli . He speculated that there would be many uses for such transducing phage, including “genetic engineering” ( Lobban 1969 , 1972 ). However, rather than directly pursuing the construction of a λ phage transducing virus as proposed, Lobban decided it would be better to focus initially on developing an in vitro DNA joining protocol to form circular dimers of phage P22 DNA from P22 DNA monomers ( Lobban 1972 ; Lobban and Kaiser 1973 ). His reasoning was that the latter was a better model system for working out the detail methodology since P22 phage DNA naturally has blunt ends and is circularly permuted and, therefore, would be unable to dimerize without the addition of (dA) n and (dT) n tails.

During this period, Lobban and Jackson were in close communication, freely sharing enzymes and their findings while they worked on their respective projects. Unbeknownst to them, Jensen et al. (1971) were also attempting to join together two DNAs in vitro by synthesizing complementary tails with TdT followed by incubation with DNA ligase in the presence of DNA polymerase I; in this case, they used phage T7 DNA as the two templates. Clearly, the idea of joining together DNAs by generating cohesive ends with TdT was a logical extension of facts already known to many biochemists at this time.

A suitable DNA for linking to SV40 DNA was developed during the winter of 1971 through the collaborative efforts of D. Berg 7 et al .(1974). This DNA, called λ dvgal 120, contains both the genes from phage λ necessary for replication as an autonomous plasmid in E. coli and an intact gal operon, i.e ., the three genes from E. coli needed for metabolizing galactose. At the time, Mertz also showed that purified λ dvgal 120 DNA could be reestablished as an autonomously replicating plasmid in E. coli using a procedure originally developed by Mandel and Higa (1970) for transformation of linear phage DNAs. Thus, both the mammalian and bacterial cloning DNAs were in hand, along with methods for reintroducing them into their host cells.

Several kinds of experiments could potentially be explored with an SV40-λ dvgal 120 recombinant DNA. One was to determine whether the E. coli gal operon is expressed in mammalian cells and, if so, to study its expression and regulation in that environment. The other objective was to determine whether the SV40-λ dvgal 120 plasmid DNA could replicate autonomously in E. coli . The latter would provide a way (i) to produce large quantities of SV40 DNA and, possibly, its encoded proteins, and (ii) to generate mutants of SV40 in vitro or in vivo that could be propagated in E. coli and their phenotypes assessed by introduction into mammalian cells.

Creating recombinant DNA in vitro :

Both SV40 and λ dvgal 120 exist naturally as circular DNA molecules. Thus, as a first step, methods were needed to cleave each of them once to produce full-length linear molecules. This task was achieved in two ways. One procedure relied on the fact that circular DNAs can be cleaved to linear molecules by incubation with pancreatic DNase I in the presence of the divalent cation Mn 2+ , a condition that limits the reaction to one or two double-stranded cleavages per molecule ( Melgar and Goldthwait 1968 ). The second procedure grew out of the seminal findings of Kelly and Smith (1970) and Danna and Nathans (1971) that some restriction endonucleases can be used to quantitatively cleave DNAs at unique sites. By testing several DNA restriction enzymes, John Morrow 8 found one, Eco RI endonuclease, an enzyme from E. coli discovered by Herbert Boyer, 9 that cleaved both SV40 ( Morrow and Berg 1972 ) and λ dvgal 120 (D. Berg et al. 1974 ) DNA once at unique sites. The latter method was chosen for our studies because it generated much higher yields of linear DNAs that were both unit length and devoid of single-strand nicks.

On the basis of a finding by Lobban, Jackson and Symons pared back the 5′-ends of the duplex linear DNAs with a λ phage-encoded 5′-exonuclease to improve the TdT-catalyzed addition of nucleotides at the 3′-ends. Accordingly, they digested Eco RI-cleaved SV40 and λ dvgal 120 DNAs with λ 5′-exonuclease to create 3′-extensions and then added 50–100 adenylate nucleotides to the 3′-ends of the SV40 DNA and 50–100 thymidylate nucleotides to the 3′-ends of the λ dvgal 120 DNA ( Figure 2 ). Specific annealing conditions led to the formation of noncovalently associated chimeric circular DNA molecules. Because the (dA) n and (dT) n tails had only approximately similar lengths, there were gaps at the (dA) n :(dT) n joints. These gaps were filled in using E. coli DNA polymerase I in the presence of the four deoxynucleoside triphosphates and exonuclease III, and the joints were covalently sealed using E. coli DNA ligase I. Exo III was included in the final reaction mixture because Lobban had found that the enzyme's presence greatly increased his yield of covalently closed circular P22 dimers. Jackson proved he had succeeded in constructing covalently closed SV40-λ dvgal 120 chimeric DNA molecules in vitro by separating them from the unreacted linear DNAs by CsCl-ethidium bromide equilibrium centrifugation and documenting their existence and size by electron microscopy ( Jackson et al. 1972 ).

Method used by Jackson et al. (1972) for constructing SV40-λ dvgal 120 recombinant DNA in vitro.

Thus, by the spring of 1972, the first chimeric recombinant DNA had been produced by sequentially using six enzymes with previously known properties: Eco RI endonuclease provided by Boyer and the others provided by colleagues in the Stanford Biochemistry Department. Undoubtedly, the ready availability of all of the above-mentioned enzymes and the expertise in their use was a very important contributor to the venture's success. Noteworthy is the fact that none of the individual procedures, manipulations, and reagents used to construct this recombinant DNA was novel; the novelty lay in the specific way in which they were used in combination. The procedure outlined above worked well with two relatively pure DNAs. However, the complexity of the products is problematic with mixtures of DNAs. Indeed, when David Hogness 10 and his colleagues used the (dA) n :(dT) n joining procedure to recombine random-sized fragments of Drosophila DNA with a bacterial plasmid, they ended up with a complex mixture of inseparable recombinants ( Wensink et al. 1974 ). To overcome that problem, a method was needed to enrich or, preferably, completely separate recombinants one from another.

The plan to construct SV40-λ dvgal 120 recombinant DNAs and to propagate them in E. coli became public in July, 1971, while Mertz was taking a course on animal cells and viruses at the Cold Spring Harbor Laboratory. Upon hearing her description of this project, Robert Pollack, the course's instructor, expressed concern about it. His anxiety, soon repeated by others, centered on the facts that: (i) SV40 can promote oncogenic transformation of human cells in culture and produce tumors in rodents; and (ii) E. coli , the presumptive carrier of the recombinant plasmid, is a natural inhabitant of the human intestinal tract. Most of the scenarios imagined the inadvertent or intentional release of E. coli carrying the SV40 DNA, with the attendant potential to spread a cancer-causing gene within the human population. Our initial reaction was that those fears were overblown and that procedures could be designed to mitigate against those risks. While some experienced tumor virologists and bacteriologists were also dismissive of the fears of the potential hazards, others thought the likelihood of something amiss happening were quite small, but not absolutely zero. Although there was little reason to believe that the SV40-λ dvgal 120 recombinant DNA itself posed a risk to human health, we, nevertheless, agreed after considerable hesitation to defer the introduction of this chimeric DNA into E. coli until better assessments regarding its safety were developed.

Prompted by concerns relating to the possible oncogenic potential of SV40 in humans, Berg and other prominent scientists convened a meeting to assess the risks of working with tumor viruses and recombinant DNAs that contain them. That meeting, sponsored by the National Institutes of Health and the National Science Foundation, was held in January, 1973 at the Asilomar Conference Center in Pacific Grove, California. Although no well-documented problems arising from working with these agents were uncovered, several recommendations were made for scientists working with them ( Hellman et al. 1973 ). These recommendations included to periodically monitor researchers who work with tumor viruses for infection, to prohibit pipetting by mouth, and to use laminar flow hoods during all manipulations involving potentially infectious material.

Shortly thereafter, another important breakthrough occurred. In the spring of 1972, Mertz discovered an unexpected property of the Eco RI endonuclease. She had repeatedly observed that Eco RI-cleaved linear SV40 DNA is approximately one-tenth as infectious as circular SV40 DNA in monkey cells; the recovered replicated viral DNA is circular and contains an intact Eco RI site. Although Kelly and Smith (1970) had shown that the restriction endonuclease they had characterized from Haemophilus influenza cleaves DNA leaving blunt ends, Mertz hypothesized that Eco RI-cut SV40 DNA contained cohesive ends and that it could form circles by annealing of these ends in the same way that linear phage λ DNA forms circles. Using electron microscopy, she showed that incubation of Eco RI-cut linear SV40 DNA with E. coli DNA ligase I at 15° results in the efficient reformation of covalently closed circular DNA molecules. Then, working in collaboration with Ronald Davis, 11 Mertz determined that, although less than 1% of Eco RI-cut SV40 DNA molecules are circular when spread in 50% formamide at room temperature, more than half of them are circular when incubated and spread at 3°. Thus, the ends created by cleavage with Eco RI endonuclease are cohesive. The T m for the circular-to-linear molecule transition is 6°. Mertz and Davis (1972) also found that at least 18 of the 19 fragments of various lengths produced by Eco RI cleavage of an ∼74-kbp plasmid, F8 (P17), can form intramolecular circles when incubated and spread for electron microscopy at 3°. Thus, they concluded that all ends created by Eco RI cleavage are probably identical, cohesive, and can be joined together with DNA ligase.

To demonstrate directly that the cohesive ends created by Eco RI cleavage could be used to create chimeric DNAs, they also incubated Eco RI-cleaved SV40 DNA and Eco RI-cleaved λ dvgal 120 DNA together in equimolar amounts at high DNA concentration with E. coli DNA ligase I at 15°. While the linear DNAs ligated separately had distinctive buoyant densities in CsCl, most of the molecules produced when the two DNAs were ligated in the same reaction mixture had an intermediate buoyant density. Taken together, these experiments definitively established that any two DNA molecules whose ends are created by cleavage with Eco RI endonuclease can be readily joined together by ligation in vitro . Electron microscopic analysis of the lengths of these chimeric DNA molecules indicated that most consisted of circular DNAs containing a mixture of three or more copies of the input DNAs. Thus, the products of this reaction probably included some containing two or more tandem copies of λ dvgal 120 DNA covalently linked to one or more copies of SV40 DNA. These chimeric molecules would have been able to replicate in E. coli. However, that supposition was not tested because of our self-imposed moratorium on producing E. coli containing SV40 oncogenes.

Boyer was promptly informed about the discovery that cleavage of DNA with Eco RI endonuclease generates cohesive ends. Together with Joe Hedgpeth 12 and Howard Goodman, 13 Boyer used this knowledge to determine that the nucleotide sequence of the 5′-extensions generated by cleavage with Eco RI endonuclease is 5′-AATT-3′ ( Hedgpeth et al. 1972 ). This finding agreed well with the Mertz and Davis (1972) estimate of 4 or 6 bases obtained by measuring the T m for annealing of the ends.

Cloning in bacteria:

Prior to 1972, Stanley Cohen 14 had been studying the structure and replication of DNA plasmids such as pSC101 that bear antibiotic resistance genes in bacteria. Aware of the not-yet-published findings of Mertz and Davis (1972) and D. Berg et al . (1974) , Cohen realized that these techniques could be quite helpful for his research. In collaboration with Annie Chang 15 and Leslie Hsu, 15 Cohen showed that Eco RI endonuclease-cleaved pSC101 DNA can be taken up by E. coli where it recircularizes and replicates as an autonomously replicating plasmid ( Cohen et al. 1972 ). Next, Cohen, Chang, Boyer, and Robert Helling 16 (1973) relied on the cohesive property of Eco RI endonuclease-generated ends to recombine pSC101 with a segment of DNA from an E. coli plasmid that contained a different antibiotic resistance gene; the new plasmid could be propagated in E. coli where it expressed both antibiotic resistance properties. Chang and Cohen (1974) then constructed a wholly novel interspecies recombinant plasmid by joining together pSC101 and a plasmid DNA originating from the gram-positive bacterium, Staphylococcus aureus. This chimeric plasmid propagated efficiently in gram-negative E. coli , exhibiting the unique antibiotic resistance characteristics of both parental plasmids. Thus, Cohen and his collaborators demonstrated that novel recombinant DNAs created in vitro , including even interspecies ones, can be cloned, propagated, and expressed in E. coli .

The finding that DNAs of different microbial origins can be propagated in E. coli still left unanswered the provocative, key question of whether eukaryotic or, for that matter, any DNA can be cloned in a bacterial host. John Morrow, who was finishing his Ph.D. thesis research in 1973 in Berg's laboratory and was aware of the Mertz and Davis (1972) and Cohen et al. (1972 , 1973 ) discoveries, undertook to answer that question. Knowing about the concerns of introducing potentially biohazardous genes into bacteria, Morrow proposed to Boyer at the June 1973 Gordon Conference on Nucleic Acids that they attempt to propagate Xenopus laevis ribosomal DNA in E. coli . Morrow had already determined that a sample of purified X. laevis ribosomal DNA obtained from Donald Brown, Morrow's prospective postdoctoral mentor, was cleaved by Eco RI endonuclease. With Cohen joining the collaborative effort, pSC101 was chosen as the cloning vector because it contained a readily selectable marker. After ligating the mixture of Eco RI-cleaved pSC101 and X. laevis ribosomal DNAs, they selected and characterized clones expressing the pSC101-encoded antibiotic resistance gene. The outcome was quite clear: ∼20% of the bacterial clones containing pSC101 DNA also contained 18S or 28S X. laevis ribosomal DNA ( Morrow et al. 1974 ). In some instances, RNA complementary to the X. laevis ribosomal DNA could be detected in the cells containing the chimeric plasmid DNAs, although these RNAs probably arose from transcripts initiated within pSC101 sequences. Thus, the Morrow et al. experiment demonstrated that genes from a eukaryotic organism can be cloned and replicated in E. coli .

The profound implication of this experiment was that DNA from any organism on the planet could probably be cloned and propagated in E. coli . This experiment also provided a prototype for many subsequent ones aimed at cloning specific genes. By 1976, Davis and his colleagues demonstrated functional expression of a protein-coding gene from yeast ( Struhl 2008 ). Eventually, cloning served as the archetypical approach used to sequence entire genomes. It also paved the way toward creating E. coli containing recombinant plasmids in which genes encoding proteins or RNAs are linked to regulatory sequences, thereby enabling the expression of their products.

Patenting and start of biotechnology industry:

None of the members of the Berg, Kaiser, or Davis groups ever considered patenting the reagents or procedures that were used for recombining DNA in vitro . Neither had the scientists who discovered TdT, DNA polymerases, DNA ligases, exonucleases, and restriction enzymes ever sought patents for their efforts. Indeed, few, if any, of the discoveries, reagents, and methods that constitute the foundations of molecular biology were ever patented. While some academic institutions such as the University of Wisconsin–Madison had a long history of patenting inventions in the biological and biochemical sciences ( e.g. , vitamins, antibiotics), the sociology among most U. S. life scientists prior to the 1970s was to eschew patents, believing that they would restrict the free flow of information and reagents and impede the pace of discovery. However, that reticence disappeared in November, 1974 when Stanford University and the University of California at San Francisco jointly filed a United States patent application citing their respective faculty members, Stanley Cohen and Herbert Boyer, as the sole inventors of the recombinant DNA technology. Their claims to commercial ownership of the techniques for cloning all possible DNAs, in all possible vectors, joined in all possible ways, in all possible organisms were dubious, presumptuous, and hubristic. Nevertheless, these claims, only slightly modified, were eventually approved in 1980 by the U. S. Patent Office ( Cohen and Boyer 1980 ). By employing what proved to be very wise terms regarding licensing and royalties, the two universities collectively garnered nearly $300 million in revenues during the life of this and two other related patents. Following university practices, Cohen, Boyer, and their respective university departments each received shares of the income from the “Cohen-Boyer patents,” while the institutions' shares were used to support universitywide research and education. In retrospect, Stanford's and UCSF's action set in motion an escalating cascade of patent claims by universities covering their faculties' respective discoveries that continues to this day. The emergence of the biotechnology industry followed naturally from the encouragement of academic scientists to patent their research discoveries and to explore their newly discovered entrepreneurial instincts. The early successes of Genentech, Biogen, and Amgen owe much to those encouragements. The events leading to the approval of the Cohen-Boyer patents and the founding of the biotechnology industry are described in detail by Hughes (2001) and Yi (2008) .

Development of regulatory guidelines:

Boyer's presentation of the Cohen et al. (1973) experiments, resulting in the creation of plasmids with novel combinations of antibiotic resistance genes, triggered concerns about the safety of such recombinants among the participants attending the June 1973 Gordon Conference on Nucleic Acids ( Singer and Söll 1973 ). In response to those concerns, the U. S. National Academy of Sciences (NAS) asked Berg to convene a committee of scientists who were familiar with and likely to use the new tools in their own research. That committee was asked to examine the scientific prospects and potential risks of what came to be known as recombinant DNA. Just before the committee met, news of the Morrow et al. (1974) experiment became known. Even though this experiment involved the cloning of a DNA segment generally accepted as being quite innocuous, its success was viewed as having “opened the door” to cloning DNAs from any biological source, including viruses, toxin-coding genes, and mammalian oncogenes. At the spring 1974 meeting of the NAS committee, the participants acknowledged that recombinant DNA technology had great promise for advancing basic and applied biology, but agreed there was insufficient information and data to determine the magnitude, if any, of the risks (P. Berg et al. 1974 ). In light of the uncertainty, the committee recommended that certain types of DNA cloning experiments be deferred until a conference of experts could be convened to assess the nature of the benefits and risks associated with such research.

The International Conference on Recombinant DNA was convened in February of 1975 at the Asilomar Conference Center in Pacific Grove, California. After considerable debate, the conference recommended that the moratorium on the previously deferred experiments be lifted and replaced with guidelines governing such research ( Berg et al. 1975 ). In the summer of 1976, the National Institutes of Health issued its first set of Guidelines for Research Involving Recombinant DNA . These guidelines and analogous ones from other international jurisdictions along with their updates have been adhered to throughout the world. In the over three decades since adoption of these various regulations for conducting recombinant DNA research, many millions of experiments have been performed without reported incident. No documented hazard to public health has ever been attributable to the applications of recombinant DNA technology. Moreover, the concern that moving DNA among species would breach customary breeding barriers with profound effects on natural evolutionary processes has substantially diminished as research has revealed such exchanges occur in nature as well. Table 1 summarizes the chronology as we know it of the events described in this essay.

Chronology of main events relating to development of methods for constructing and cloning recombinant DNAs

Year(s) in which event occurred.

Impacts of recombinant DNA technology:

The most far-reaching consequence of the emergence of the recombinant DNA technology has been the great strides made in understanding fundamental life processes and the ability to investigate problems that had previously been unapproachable. Emerging from myriad investigations has been the appreciation that nothing in the man-made world rivals the complexity and diversity of this earth's organisms. No man-made information system invented to date comes anywhere close to containing the amount of information encoded in their genomes or encompassing the complexity of the intricate machinery for their functioning. We have learned enough to reveal how much we do not know and to acknowledge that nature's secrets are not beyond our capabilities of discovery.

The advances made possible by recombinant DNA technology have profound implications for the future of medicine for they have placed us at the threshold of new methods of diagnosis, prevention, and treatment of numerous human diseases. Hormones, vaccines, therapeutic agents, and diagnostic tools developed using recombinant DNA methods are already greatly enhancing medical practice. Although the production and consumption of genetically engineered food are realities, the benefits have yet to be fully realized. Nevertheless, recombinant DNA technologies will, undoubtedly, play roles in the future in increasing the supply of both food and energy needed by the world's growing human population.

This article is dedicated to Arthur Kornberg, who fostered a group of colleagues that made this work possible.

Paul Berg was professor and chair of the Biochemistry Department at Stanford University Medical Center at the time of the events described here.

Janet Mertz was a graduate student in P. Berg's laboratory from 1970 to 1975.

David Jackson was a postdoctoral fellow in P. Berg's laboratory.

Robert Symons was a visiting professor in P. Berg's laboratory.

Peter Lobban was a graduate student in A. D. Kaiser's laboratory in the Biochemistry Department at Stanford University.

Douglas Berg was a postdoctoral fellow in A. D. Kaiser's laboratory.

John Morrow was a graduate student in P. Berg's laboratory.

Herbert Boyer was an associate professor in the Department of Microbiology at University of California, San Francisco (UCSF).

David Hogness was a professor in the Biochemistry Department at Stanford University.

Ronald Davis was an assistant professor in the Biochemistry Department at Stanford University.

Joe Hedgpeth was a postdoctoral fellow in Boyer's laboratory.

Howard Goodman was an associate professor in the Department of Biochemistry and Biophysics at UCSF.

Stanley Cohen was an assistant professor in the Department of Medicine at Stanford University.

Annie Chang and Leslie Hsu were technician and graduate student, respectively, in Cohen's laboratory.

Robert Helling was a postdoctoral fellow in Boyer's laboratory.

We thank Douglas Berg, William Dove, David Jackson, A. Dale Kaiser, Peter Lobban, John Morrow, Maxine Singer, and Adam Wilkins for their suggestions for improving this article and Peter Lobban for permission to reproduce Figure 1 . Much of the work described here was funded in large part by grants to Paul Berg from the National Institutes of Health and the American Cancer Society.

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The Recombinant DNA Technology Era

• First Online: 27 April 2022

Cite this chapter

• Manisha Modak 4 ,
• Narendra Nyayanit 4 ,
• Aruna Sivaram   ORCID: orcid.org/0000-0003-4942-4114 5 &
• Nayana Patil   ORCID: orcid.org/0000-0002-8743-4578 5  

Part of the book series: Techniques in Life Science and Biomedicine for the Non-Expert ((TLSBNE))

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Humans have been practicing biotechnology since a long time ago to prepare fermented food and beverages and to treat diseases. Onset of the microscopic era in the seventeenth century gave a momentum to the use of microbes in various applications. With rapid evolution of technologies, today biotechnology has become an indispensable part of various industries. One of the most important developments in biotechnology has been the concept of recombinant DNA, where organisms can be genetically modified to suit the requirements. Several DNA editing tools and methods have evolved to precisely control the manipulation of the genome in any living organism, simplify the tedious procedures, making it faster and cheaper. In this chapter, we will discuss the fascinating progress that has been made in this technology over the past centuries.

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A Brief Introduction to Recombinant DNA Technology

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Modak, M., Nyayanit, N., Sivaram, A., Patil, N. (2022). The Recombinant DNA Technology Era. In: A Complete Guide to Gene Cloning: From Basic to Advanced . Techniques in Life Science and Biomedicine for the Non-Expert. Springer, Cham. https://doi.org/10.1007/978-3-030-96851-9_1

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People have known since time immemorial that it is possible to breed animals and plants to enhance their desirable characteristics and to ‘breed out’ their undesirable traits. And they have argued that, since it is possible to breed horses for speed or stamina, it ought to be possible to breed human beings for specifically human qualities such as intelligence and kindness and sociability.

Ethical and moral arguments are based on the fact that in spite of its benefits and advantages, recombinant DNA technology hides many threats for community and future generations.

The main ethical issue of recombinant DNA technology concerns the nature of investigations and their impact on future generations. Recombinant DNA is an artificial DNC created by two or more DNA strands that would not usually occur together in natural environment. “Recombinant DNA technology is thus a process whereby genetic material is manipulated in order to develop biological compounds. It is here where industrial capitalization is the greatest and popular imagination fixates” (Pepa 1998, p. 416). For if to settle the moral question about recombinant DNA technology is not thereby to settle the legal question as to whether and how recombinant DNA technology should be controlled and regulated, so also to settle the legal question is not thereby to settle the moral question. In other words, society must resist the idea that if the law is silent about a given area then ‘anything goes’ in that area (Dadachanji 2001).

There are many kinds of behavior that people may consider immoral or objectionable or undesirable, but which are not the business of the law. In a liberal democratic and pluralist society the law is not really concerned with the enforcement of morality but rather with providing a framework of peace and order within which people may exercise their personal liberty to the greatest possible extent and make their own personal moral choices and engage in their own ‘experiments in living’. The nature of recombinant DNA suggests that it would be difficult if not impossible to control all experiments and laboratories dealing with this technology. In these circumstances, scientists can create monsters or creatures (animals and even human beings). These results violate human and animal rights, freedoms and social protection from exploitation (Hanson, 1997).

The main moral argument against recombinant DNA technology is that these researchers can work with human genes and DNAs. Sixty thousand of those appear within genes, and so are one source of our biological individuality, including the diseases to which we are susceptible, and our idiosyncratic responses to medications. Those same genetic differences also provide the raw material for the genetic fingerprinting that now helps convict rapists and murderers, free wrongly convicted prisoners, and identify the victims of political “disappearances” and “ethnic cleansings” (McKelvey 2000). As important as knowledge of the human genome is, it is incomplete. True, researchers are currently filling in details missed in the first draft, chromosome by chromosome (Navidi and Arnheim1999).

But several critics of the project have pointed out that the genome is more like a list of parts than, as it has been described, the instruction book for creating a human being. A modern jetliner has about the same number of parts—100,000—as humans have proteins. But it’s a long way from those parts to a working airplane, and an even longer one from a list of our genes to a newborn baby. It will take many decades of work by thousands of researchers to identify all our genes and their controlling elements, map genes into proteins, and determine the structure and functions of those proteins. Hence the current focus on “proteomics.” The ultimate goal—mapping, modeling, and controlling the incredibly complex interactions of the network of genes and gene products over the course of development and in response to the environment—is just visible on the scientific field (McKelvey 2000).

It may then very well be the case that some of the practices and procedures in the area of biotechnology are held to be immoral or unethical by many people, but that nevertheless they are not made illegal or subject to legal control. To be made illegal it has to be shown not just that they are unethical or raise social problems but in addition that they are likely to have harmful implications for others, that is, violate people’s rights in some clear and obvious way (Notes on Moral Theology Ethical 1999). All the most recent work in genetics has shown how extraordinarily complex the genetic control and regulation of human characteristics and functions are, and how impractical it is to manipulate most of the genetic mechanisms in any direct way. Some human characteristics and pathological conditions are controlled by a single gene and these are mostly manipulable, but many others are regulated by a number of genes interacting with each other in very complex ways (Neuwald and Lawrence 1999). Some genes directly determine specific human characteristics, but others provide conditions or dispositions for human traits and functions (Pepa, 1998). Again, there is a continual reaction between genetic factors and external environmental factors. What this means is that, while it is quite feasible to predict that a number of single gene-based diseases will be able to be remedied by genetic manipulation, positive eugenics or the reshaping of human beings is, scientifically speaking, likely to remain an idle dream (Pollack 2006).

In contrast to these views, some critics admit that recombinant DNA technology proposes great opportunities for medicine to treat incurable diseases and create new species. They state that society needs as many groups in the community as possible keeping watch on developments in biotechnology, raising questions, initiating discussion, issuing reports. The community as a whole needs to educate itself so that it can deal in a positive way with the possibilities disclosed by the new biotechnology. “Transgenically-created animals function as living test tubes that permit scientists to imitate human diseases, attempt better treatments, and produce larger amounts of beneficial proteins more cheaply than ever before” (Pepa 1998, p. 416). In particular the media has a central function here, avoiding sensationalism and what critics call the science fiction approach and trying to promote a genuine and responsible public debate on what are really matters of life and death. In my opinion that debate is of the greatest importance and my hope is that these six lectures may have contributed to it. Anyone who knows something about the dehumanising, effects of some genetic diseases would want animals and plats to escape them if it were possible. It’s not a matter of wanting to have a ‘perfect’ animal made to order (Pollack 2006).

In sum, recombinant DNA technology hides many threats but proposes great opportunities for the society to overcome incurable diseases and create new plants. Recombinant DNA technology should be based on strong moral principles and rules in order to prevent violation of human and animal rights and prevent misconduct of researchers working with this technology. It needs also to be shown that the prohibitions of the law are likely to be obeyed by the generality of people and that enforcement of the law will not bring about more harm than good in a society where there is a plurality of widely differing moral views and convictions. This disjunction between the sphere of law and the sphere of morality cuts both ways.

Bibliography

Dadachanji, D.K. September 2001, Unraveling the Human Thread of Life. World and I , 16, p. 136.

Hanson, M.J. 1997, Religious Voices in Biotechnology: The Case of Gene Patenting. The Hastings Center Report, 27, p. 1.

Navidi, W., Arnheim, N. 1999, Combining Data from Polymerase Chain Reaction DNA Typing Experiments: Application to Sperm Typing Data. Jo urnal of the American Statistical Association , 94, 726-729.

Neuwald, A.E., Lawrence, Ch. E. 1999, Markovian Structures in Biological Sequence Alignments. Journal of the American Statistical Association , 94 (445), p. 1.

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Recombinant DNA technology and DNA sequencing

Affiliation.

• 1 Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdon.
• PMID: 31652313
• DOI: 10.1042/EBC20180039

DNA present in all our cells acts as a template by which cells are built. The human genome project, reading the code of the DNA within our cells, completed in 2003, is undoubtedly one of the great achievements of modern bioscience. Our ability to achieve this and to further understand and manipulate DNA has been tightly linked to our understanding of the bacterial and viral world. Outside of the science, the ability to understand and manipulate this code has far-reaching implications for society. In this article, we explore some of the basic techniques that enable us to read, copy and manipulate DNA sequences alongside a brief consideration of some of the implications for society.

Keywords: CRISPR; DNA sequencing; biochemical techniques and resources.

© 2019 The Author(s).

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AP®︎/College Biology

Course: ap®︎/college biology   >   unit 6.

• Introduction to genetic engineering

Intro to biotechnology

• DNA cloning and recombinant DNA
• Overview: DNA cloning
• Polymerase chain reaction (PCR)
• Gel electrophoresis
• DNA sequencing
• Applications of DNA technologies
• Biotechnology

Key points:

• Biotechnology is the use of an organism, or a component of an organism or other biological system, to make a product or process.
• Many forms of modern biotechnology rely on DNA technology.
• DNA technology is the sequencing, analysis, and cutting-and-pasting of DNA.
• Common forms of DNA technology include DNA sequencing , polymerase chain reaction , DNA cloning , and gel electrophoresis .
• Biotechnology inventions can raise new practical concerns and ethical questions that must be addressed with informed input from all of society.

Introduction

What is biotechnology.

• Beer brewing . In beer brewing, tiny fungi (yeasts) are introduced into a solution of malted barley sugar, which they busily metabolize through a process called fermentation. The by-product of the fermentation is the alcohol that’s found in beer. Here, we see an organism – the yeast – being used to make a product for human consumption.
• Penicillin. The antibiotic penicillin is generated by certain molds. To make small amounts of penicillin for use in early clinical trials, researchers had to grow up to 500 ‍   liters of “mold juice” a week 1 ‍   . The process has since been improved for industrial production, with use of higher-producing mold strains and better culture conditions to increase yield 2 ‍   . Here, we see an organism (mold) being used to make a product for human use – in this case, an antibiotic to treat bacterial infections.
• Gene therapy. Gene therapy is an emerging technique used to treat genetic disorders that are caused by a nonfunctional gene. It works by delivering the “missing” gene’s DNA to the cells of the body. For instance, in the genetic disorder cystic fibrosis, people lack function of a gene for a chloride channel produced in the lungs. In a recent gene therapy clinical trial, a copy of the functional gene was inserted into a circular DNA molecule called a plasmid and delivered to patients’ lung cells in spheres of membrane (in the form of a spray) 3 ‍   . In this example, biological components from different sources (a gene from humans, a plasmid originally from bacteria) were combined to make a new product that helped preserve lung function in cystic fibrosis patients.

What is DNA technology?

Examples of dna technologies.

• DNA cloning. In DNA cloning , researchers “clone” – make many copies of – a DNA fragment of interest, such as a gene. In many cases, DNA cloning involves inserting a target gene into a circular DNA molecule called a plasmid. The plasmid can be replicated in bacteria, making many copies of the gene of interest. In some cases, the gene is also expressed in the bacteria, making a protein (such as the insulin used by diabetics).
• Polymerase chain reaction (PCR). Polymerase chain reaction is another widely used DNA manipulation technique, one with applications in almost every area of modern biology. PCR reactions produce many copies of a target DNA sequence starting from a piece of template DNA. This technique can be used to make many copies of DNA that is present in trace amounts (e.g., in a droplet of blood at a crime scene).
• Gel electrophoresis. Gel electrophoresis is a technique used to visualize (directly see) DNA fragments. For instance, researchers can analyze the results of a PCR reaction by examining the DNA fragments it produces on a gel. Gel electrophoresis separates DNA fragments based on their size, and the fragments are stained with a dye so the researcher can see them. Based on similar diagram in Reece et al. 5 ‍  
• DNA sequencing. DNA sequencing involves determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a DNA molecule. In some cases, just one piece of DNA is sequenced at a time, while in other cases, a large collection of DNA fragments (such as those from an entire genome) may be sequenced as a group. What is a genome? A genome refers to all of an organism's DNA. In eukaryotes, which have a nucleus in their cells to hold their DNA, the word genome is usually used for the nuclear genome (DNA found in the nucleus), excluding the DNA found in organelles such as chloroplasts or mitochondria.

Biotechnology raises new ethical questions

• Some of these relate to privacy and non-discrimination. For instance should your health insurance company be able to charge you more if you have a gene variant that makes you likely to develop a disease? How would you feel if your school or employer had access to your genome?
• Other questions relate to the safety, health effects, or ecological impacts of biotechnologies. For example, crops genetically engineered to make their own insecticide reduce the need for chemical spraying, but also raise concerns about plants escaping into the wild or interbreeding with local populations (potentially causing unintended ecological consequences).
• Biotechnology may provide knowledge that creates hard dilemmas for individuals. For example, a couple may learn via prenatal testing that their fetus has a genetic disorder. Similarly, a person who has her genome sequenced for the sake of curiosity may learn that she is going to develop an incurable, late-onset genetic disease, such as Huntington's.

Educate yourself and share your perspective

Works cited:.

• American Chemical Society. (2016). Discovery and development of penicillin. In Chemical landmarks . Retrieved from http://www.acs.org/content/acs/en/education/whatischemistry/landmarks/flemingpenicillin.html .
• Meštrović, T. and Chow, S. (2015, April 29). Penicillin production. In News medical . Retrieved from http://www.news-medical.net/health/Penicillin-Production.aspx .
• Alton, E. W. F. W., Armstrong, D. K., Ashby, D., Bayfield, K. J., Bilton, Diana, Bloomfield, E. V., ... Wolstenholme-Hogg, P. (2015). Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: A randomised, double-blind, placebo-controlled, phase 2b trial. Lancet Respiratory Medicine , 3 (9), 684-691. http://dx.doi.org/10.1016/S2213-2600(15)00245-3 .
• Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., and Jackson, R. B. (2011). The DNA toolbox. In Campbell biology (10th ed., pp. 408-409). San Francisco, CA: Pearson.
• Reece, J. B., Taylor, M. R., Simon, E. J., and Dickey, J. L. (2012). Figure 12.13. Gel electrophoresis of DNA. In Campbell biology: Concepts & connections (7th ed., p. 243).

Additional references:

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Biotechnology - Recombinant DNA

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