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    what process involves the introduction of recombinant dna plasmids into a bacterial colony?

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    Recombinant Plasmid

    Recombinant Plasmid

    The recombinant plasmid molecule that consists of foreign DNA and vector-specific sequence is introduced into bacterial host cells, usually a strain of Escherichia coli, by the process of transformation.

    From: Foundations of Anesthesia (Second Edition), 2006

    Related terms:

    AntigenEnzymeProteinDNAPhosphoproteinPlasmidPlasmid DNAEscherichia coliBacterium

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    Techniques for Nucleic Acid Engineering

    Şükrü Tüzmen, ... Candan Hızel, in Omics Technologies and Bio-Engineering, 2018

    14.5 Recombinant DNA Techniques

    Recombinant DNA technology was made possible through the discovery, and application of restriction enzymes for which Werner Arber, Daniel Nathans, and Hamilton Smith received the 1978 Nobel Prize in Medicine [Physiology or Medicine 1978—Press Release].

    With the recent advances in molecular biology, it is now possible to mix genetic material from multiple organisms together (molecular cloning) to create DNA sequences that are otherwise not found naturally in biological organisms. Techniques like molecular cloning are used to create these rDNA molecules in the laboratories. Vectors are used to transfer and express these foreign rDNA fragments in suitable host organisms such as bacteria. R-DNA technology facilitated a whole new world in scientific research. R-DNA technology employs palindromic sequences, and results in the creation of blunt and sticky (staggered) ends (Fig. 14.22). Since its development, many organisms and food products have been genetically modified. Utilizing rDNA technology and synthetic DNA molecules, literally any DNA sequence can be created and inserted into any of a very broad range of living organisms. Thus, it is not surprising that this topic also has many controversies attached to it.

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    Figure 14.22. Recombinant plasmid formation.

    Adapted from http://en.wikipedia.org/wiki/Recombinant_DNA.

    14.5.1 Creation of Recombinant (Artificial) DNA

    Recombinant plasmid formation involves construction of rDNA, in which a foreign DNA fragment is inserted into a plasmid vector. The gene indicated by white color in Fig. 14.22 is inactivated upon insertion of the foreign DNA fragment illustrated by jigsaw pieces (Fig. 14.22).

    14.5.2 Chimeric/rDNA

    Recombinant DNA molecules are occasionally referred to as chimeric DNA, because they are usually constructed using materials from two different species. The term “molecular cloning” is used to indicate the laboratory process utilized to make rDNA (Campbell and Reece, 2002; Walter et al., 2008; Berg et al., 2010; Watson, 2007) (Fig. 14.23).

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    Figure 14.23. Gene cloning.

    Adapted from http://en.wikipedia.org/wiki/Recombinant_DNA.

    14.5.2.1 Steps of Cloning DNA Fragments (Gene Cloning) to Create rDNA

    Step 1. Small circular DNA molecules (plasmids) are removed from bacteria. These plasmids serve as vectors (molecules to carry genes of interest).

    Step 2. DNA containing the gene of interest to be cloned is isolated from particular cells/tissues.

    Step 3. Unique single site recognizing restriction endonucleases are utilized, which recognize specific restriction site(s) (short sequences of 4–8 bp long).

    Step 4. These unique restriction endonuclease are used to cut both the plasmid DNA (vector) and the DNA to be cloned (insert) into the plasmid vector, creating either overhangs called sticky ends or filled ends called blunt ends.

    Step 5. Following the restriction enzyme, digestion of both the vector and the insert, then the digested vector and insert fragments, whether with sticky ends or blunt ends, are joined together via complementary base pairing, via a DNA ligase enzyme. The gene of interest gets included into some of the plasmids forming recombinant plasmids. Other plasmids close right back up, remaining unchanged (without an insert).

    Step 6. DNA ligase enzyme makes the bonds permanent between complementarily paired bases, by attaching nucleotides to each other with phosphodiester bonds.

    Step 7. The next experiment is to mix the plasmids from Step 6 with competent bacteria. This process is called transformation. During transformation some of the bacteria take up the plasmids. Here bacteria are utilized to clone (multiply in number) the rDNA.

    Step 8. In some transformation experiments, a color-processing gene such as LacZ gene is utilized for confirmation of the molecular cloning (inserting a DNA fragment of interest into a plasmid vector). Plasmids with an uninterrupted LacZ gene turn their bacteria blue. In the recombinant plasmids, the inserted gene interrupts the LacZ gene, and the bacteria remain their original color (white). The bacteria that did not take up any plasmid DNA also remain uncolored/white.

    Step 9. For selection against positive bacterial clones (clones that have taken recombinant plasmids), antibiotics are added to the growth media, where the bacteria are grown. As the plasmids contain the genes for antibiotic resistance, only bacteria, which took up the plasmid, survives.

    Source : www.sciencedirect.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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