LS17DNAcloning - 1 • Allows manipulation of a specific fragment of DNA in the test tube • Reintroduced into cells and grown in large

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Unformatted text preview: 1 • Allows manipulation of a specific fragment of DNA in the test tube • Reintroduced into cells and grown in large quantities (cloned) • Any gene can be isolated, amplified, sequenced and modified for further study Lecture 17: DNA CLONNING Problems in working with DNA • DNA fragments of different genes have the similar shape and similar charge to mass ratio, so one gene cannot be easily distinguished from another like proteins. • Genes are all connected in vast DNA molecules, each gene is a very small fraction of the entire genome, so a gene can not be isolated like a protein 2 Clone :Collection of cells originated from a single cell. Collection of cells containing the identical type of DNA Collection of identical DNA fragments. ...... Collection of anything that is identical to each other and to the parent (we are not the clone of either of our parents). Cloning is to make a lot of identical copies of a DNA molecule, or a cell, or an organism. “ A gene is cloned” (= “a gene is isolated”) means that the recombinant DNA containing the target gene inserted in the vector has been identified, the recombinant DNA can be propagated indefinitely, the DNA insert (the target gene) can be cut out from the vector DNA using restriction enzyme(s) and purified away from the vector DNA whenever it is needed. CLONING Why need to isolate or clone a gene? 1. To study how genes and proteins function in the cell, and how cells function in an organism e.g. when and where is a gene expressed? Where is the protein located in the cell? how is gene expression regulated? How does a gene act to make the cell functional? How are individual genes involved in a particular disease? 2. To make therapeutical proteins (recombinant protein drugs) 3. To replace the “bad” gene in a patient (gene therapy) 4. To make improved agriculture animals and crops (GM food) 3 The best way to clone a DNA is to have a cell replicate it for us. However, a DNA fragment is generally not replicated in a cell, and it may be lost during cell division and cannot be “cloned”. In order to make multiple copies of a given DNA fragment in cells, it needs to be placed (ligated or inserted) in a vector Insert: The piece of DNA you want to amplify. Vector: a self-replicating DNA such as a plasmid or a virus that exist in multiple copies in the cell. How can we clone and amplify a gene? (1). a replicon It is the region that is essential for the replication of the plasmid. most plasmids used in DNA cloning are derivatives of the E. coli plasmids containing a ColE1 replicon. (2) a selection marker A selection marker is often a bacterial gene that confers antibiotic resistance, which is included in the vector for the purpose of easy selection, some of the often used antibiotic resistance are amp r , kan r , tet r , etc....
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This note was uploaded on 01/16/2010 for the course LIFESCI Life scien taught by Professor Lorenz,t.c. during the Fall '09 term at UCLA.

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LS17DNAcloning - 1 • Allows manipulation of a specific fragment of DNA in the test tube • Reintroduced into cells and grown in large

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