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Recombinant DNA Technology

Recombinant DNA Technology - Recombinant DNA Technology...

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1 Recombinant DNA Technology Introduction Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments. Recombinant DNA molecules are DNA fragments that are covalently linked in the lab. This technology has enabled researchers to study the relationship between genes and phenotype, and understand gene structure and function. Applications of these technologies include gene therapy and the production of transgenic plants. Gene Cloning General information Molecular biologists frequently focus on the structure and function of proteins, or the genes that encode them. Gene cloning involves the isolating and making of many copies of a gene. Table 18.1 summarizes some of the uses of gene cloning. Cloning experiments may involve two kinds of DNA molecules: chromosomal DNA and vector DNA. A vector is a small piece of DNA into which a gene of interest is introduced. The vector is then placed within a living cell, where it replicates and produces multiple identical copies of the inserted gene (Table 18.2). The vector acts as the carrier of the gene to be studied. The cell that holds the vector is called the host cell. Chromosomal DNA is usually used as the source of the gene of interest. Vectors are usually derived from plasmids. Plasmids are small circular pieces of DNA that are found in bacterial and some eukaryotic cells. Plasmids that contain genes that confer resistance to toxic compounds and antibiotics are called R factors.
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2 Plasmids contain origins of replication that are recognized by the host cell. Many plasmids contain selectable markers, which enable the cell containing the plasmid to grow in the presence of an antibiotic or other toxic substance. A viral vector is a virus that contains a chromosomal gene. Other vectors, such as cosmids, BACs, and YACs, are described later in the course. Enzymes are used to cut DNA into pieces and join the pieces together. Enzymes called restriction endonucleases, or restriction enzymes, are used to cut DNA. Restriction enzymes recognize base sequences and then cleave the DNA at two defined locations (Figure 18.1). They are found in many bacterial species, where they protect the organism from the invasion of foreign DNA, particularly bacteriophages. Examples of some restriction enzymes, their sources, and recognition sequences are provided in Table 18.3. Restriction enzymes recognize palindromic sequences. Some restriction enzymes produce fragments with “sticky ends,” which means that they will hydrogen bond with each other due to their complementary sequences. This allows for temporary (although unstable) interactions between DNA fragments. Permanent connections must be established by a DNA ligase. Gene cloning involves the insertion of DNA fragments into vectors, which are then propagated within host cells.
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