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    when a piece of dna from one organism is inserted into the genome of another through the use of recombinant plasmids, what is the end result?

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    Recombinant DNA Technology and Transgenic Animals

    “Seeing is believing with GloFish. They are absolutely stunning!” The preceding is some of the marketing material you’d read if you visited the GloFish website. Beauty may be in the eye of the beholder, but nearly everyone would agree that these first transgenic animals made available to the general public in the United States are a worthy conversation piece. But what’s the science behind them?

    Recombinant DNA Technology and Transgenic Animals

    By: Leslie Pray, Ph.D. © 2008 Nature Education

    Citation: Pray, L. (2008) Recombinant DNA technology and transgenic animals. Nature Education 1(1):51

    GloFish are the first transgenic animals available to the American public. But what's the biotechnology behind them?

    Aa Aa Aa

    Figure 1: The multicolored GloFish®.

    Courtesy of www.glofish.com. All rights reserved.

    "Seeing is believing with GloFish. They are absolutely stunning!" The preceding is some of the marketing material you'd read if you visited the GloFish website (GloFish, 2008). Beauty may be in the eye of the beholder, but nearly everyone would agree that these first—and, so far, only—transgenic animals made available to the general public in the United States (except in California, pending a formal review of their potential effect on the environment) are a worthy conversation piece. A transgenic, or genetically modified, organism is one that has been altered through recombinant DNA technology, which involves either the combining of DNA from different genomes or the insertion of foreign DNA into a genome. GloFish (Figure 1) are a type of transgenic zebrafish () that have been modified through the insertion of a green fluorescent protein () gene. Not all GloFish are green, however. Rather, there are several gene constructs, each encoding a different colored phenotype, from fluorescent yellow to fluorescent red.

    Currently, GloFish are the only recombinant-DNA animal that has been approved for human "use" by the U.S. Food and Drug Administration. Their approval has raised important questions about whether, and to what extent, genetically modified animals should be made available to consumers. But how were scientists able to create these engineered organisms in the first place? Like so many genetic technologies used today, recombinant DNA technology had its origins in the late 1960s and early 1970s. By the 1960s, scientists had already learned that cells repair DNA breaks by reuniting, or recombining, the broken pieces. Thus, it was only a matter of time before researchers identified the raw biological ingredients necessary for recombination, figured out how those ingredients functioned together, and then tried to govern the recombining process themselves.

    Early Experiments Provide the Basis for Recombinant Organisms

    Although recombinant DNA technology first emerged in the 1960s and 1970s, the basic principle of recombination had been discovered many years earlier. Indeed, in 1928, Frederick Griffith, an English medical officer studying the bacteria responsible for a pneumonia epidemic in London, first demonstrated what he termed "genetic transformation"; here, living cells took up genetic material released by other cells and became phenotypically "transformed" by the new genetic information. More than a decade later, Oswald Avery repeated Griffith's work and isolated the transforming molecule, which turned out to be DNA. These experiments showed that DNA can be transferred from one cell to another in the laboratory, thus changing the actual genetic phenotype of an organism.

    Prior to these classic experiments, the idea that the genetic material was a specific chemical that could be modified and transferred into cells was certainly controversial. But before the explosion in recombinant DNA could begin, scientists would have to learn not only how to transfer DNA, but also how to isolate and modify individual genes.

    Key Developments in Recombinant DNA Technology

    Following these early experiments, four key developments helped lead to construction of the first recombinant DNA organism (Kiermer, 2007). The first two developments revolved around how scientists learned to cut and paste pieces of DNA from different genomes using enzymes. The latter two events involved the development of techniques used to transfer foreign DNA into new host cells.

    Discovering the Cut-and-Paste Enzymes

    Figure 2

    The first major step forward in the ability to chemically modify genes occurred when American biologist Martin Gellert and his colleagues from the National Institutes of Health purified and characterized an enzyme in responsible for the actual joining, or recombining, of separate pieces of DNA (Zimmerman , 1967). They called their find "DNA-joining enzyme," and this enzyme is now known as DNA ligase. All living cells use some version of DNA ligase to "glue together" short strands of DNA during replication. Using extract, the researchers next showed that only in the presence of ligase was it possible to repair single-stranded breaks in phage DNA. (Discovered in 1950 by American microbiologist Esther Lederberg, phage is a virus particle that infects .) More specifically, they showed that the enzyme was able to form a 3'-5'-phosphodiester bond between the 5'-phosphate end of the last nucleotide on one DNA fragment and the 3'-OH end of the last nucleotide on an adjacent fragment. The identification of DNA ligase was the first of several key steps that would eventually empower scientists to attempt their own recombination experiments—experiments that involved not just recombining the DNA of a single individual, but recombining DNA from different individuals, including different species.

    Source : www.nature.com

    Overview: DNA cloning (article)

    Definition, purpose, and basic steps of DNA cloning.

    Biotechnology

    Overview: DNA cloning

    Definition, purpose, and basic steps of DNA cloning.

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    Key points:

    DNA cloning is a molecular biology technique that makes many identical copies of a piece of DNA, such as a gene.

    In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.

    The plasmid is introduced into bacteria via a process called transformation, and bacteria carrying the plasmid are selected using antibiotics.

    Bacteria with the correct plasmid are used to make more plasmid DNA or, in some cases, induced to express the gene and make protein.

    Introduction

    When you hear the word “cloning,” you may think of the cloning of whole organisms, such as Dolly the sheep. However, all it means to clone something is to make a genetically exact copy of it. In a molecular biology lab, what’s most often cloned is a gene or other small piece of DNA.

    If your friend the molecular biologist says that her “cloning” isn’t working, she's almost certainly talking about copying bits of DNA, not making the next Dolly!

    Overview of DNA cloning

    DNA cloning is the process of making multiple, identical copies of a particular piece of DNA. In a typical DNA cloning procedure, the gene or other DNA fragment of interest (perhaps a gene for a medically important human protein) is first inserted into a circular piece of DNA called a plasmid. The insertion is done using enzymes that “cut and paste” DNA, and it produces a molecule of recombinant DNA, or DNA assembled out of fragments from multiple sources.

    Diagram showing the construction of a recombinant DNA molecule. A circular piece of plasmid DNA has overhangs on its ends that match those of a gene fragment. The plasmid and gene fragment are joined together to produce a gene-containing plasmid. This gene-containing plasmid is an example of recombinant DNA, or a DNA molecule assembled from DNA from multiple sources.

    Next, the recombinant plasmid is introduced into bacteria. Bacteria carrying the plasmid are selected and grown up. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains.

    What is the point of making many copies of a DNA sequence in a plasmid? In some cases, we need lots of DNA copies to conduct experiments or build new plasmids. In other cases, the piece of DNA encodes a useful protein, and the bacteria are used as “factories” to make the protein. For instance, the human insulin gene is expressed in E. coli bacteria to make insulin used by diabetics.

    [More about insulin and diabetes]

    Steps of DNA cloning

    DNA cloning is used for many purposes. As an example, let's see how DNA cloning can be used to synthesize a protein (such as human insulin) in bacteria. The basic steps are:

    Cut open the plasmid and "paste" in the gene. This process relies on restriction enzymes (which cut DNA) and DNA ligase (which joins DNA).

    Insert the plasmid into bacteria. Use antibiotic selection to identify the bacteria that took up the plasmid.

    Grow up lots of plasmid-carrying bacteria and use them as "factories" to make the protein. Harvest the protein from the bacteria and purify it.

    Let's take a closer look at each step.

    1. Cutting and pasting DNA

    How can pieces of DNA from different sources be joined together? A common method uses two types of enzymes: restriction enzymes and DNA ligase.

    A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. If two molecules have matching overhangs, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.

    [See a diagram of restriction enzymes and DNA ligase]

    Our goal in cloning is to insert a target gene (e.g., for human insulin) into a plasmid. Using a carefully chosen restriction enzyme, we digest:

    The plasmid, which has a single cut site

    The target gene fragment, which has a cut site near each end

    Then, we combine the fragments with DNA ligase, which links them to make a recombinant plasmid containing the gene.

    Diagram depicting restriction digestion and ligation in a simplified schematic.

    We start with a circular bacterial plasmid and a target gene. On the two ends of the target gene are restriction sites, or DNA sequences recognized by a particular restriction enzyme. In the plasmid, there is also a restriction site recognized by that same enzyme, right after a promoter that will drive expression in bacteria.

    Both the plasmid and the target gene are (separately) digested with the restriction enzyme. The fragments are purified and combined. They have matching "sticky ends," or single-stranded DNA overhangs, so they can stick together.

    Source : www.khanacademy.org

    recombinant DNA

    recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet)

    recombinant DNA

    genetic engineering

    Alternate titles: recombinant DNA technology

    By Anthony J.F. Griffiths • Edit History

    DNA extraction; recombinant DNA

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    Key People: Stanley Cohen Paul Berg Mario R. Capecchi

    Related Topics: genetic engineering DNA in vitro mutagenesis gene disruption

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    Top Questions

    What is recombinant DNA technology?

    When was recombinant DNA technology invented?

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    Summary

    Read a brief summary of this topic

    recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.

    DNA cloning

    In biology a clone is a group of individual cells or organisms descended from one progenitor. This means that the members of a clone are genetically identical, because cell replication produces identical daughter cells each time. The use of the word clone has been extended to recombinant DNA technology, which has provided scientists with the ability to produce many copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. They are small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra DNA that is spliced into them.

    recombinant DNA

    Steps involved in the engineering of a recombinant DNA molecule.

    Encyclopædia Britannica, Inc.

    Source : www.britannica.com

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