E18 - E1 Sticky ends which are complementary in their DNA...

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E1. Sticky ends, which are complementary in their DNA sequence, will promote the binding of DNA fragments to each other. This binding is due to hydrogen bonding. E2. Remember that AT base pairs form two hydrogen bonds while GC base pairs form three hydrogen bonds. The order (from stickiest to least sticky) would be: Bam HI = Pst I = Sac I > Eco RI > Cla I. E3. All vectors have the ability to replicate when introduced into a living cell. This ability is due to a DNA sequence known as an origin of replication. Modern vectors also contain convenient restriction sites for the insertion of DNA fragments. These vectors also contain selectable markers, which are genes that confer some selectable advantage for the host cell that carries them. The most common selectable markers are antibiotic-resistance genes, which confer resistance to antibiotics that would normally inhibit the growth of the host cell. E4. In conventional gene cloning, many copies are made because the vector replicates to a high copy number within the cell, and the cells divide to produce many more cells. In PCR, the replication of the DNA to produce many copies is facilitated by primers, nucleotides, and Taq polymerase. E5. First, the chromosomal DNA that contains the source of the gene that you want to clone must be obtained from a cell (tissue) sample. A vector must also be obtained. The vector and chromosomal DNA are digested with a restriction enzyme. They are mixed together to allow the sticky ends of the DNA fragments to bind to each other, hopefully to create a hybrid vector. DNA ligase is then added to promote covalent bonds. The DNA is then transformed or transfected into a living cell. The vector will replicate and the cells will divide to produce a colony of cells that contain “cloned” DNA pieces. To identify colonies that contain the gene you wish to clone, you must use a probe that will specifically identify a colony containing the correct hybrid vector. A probe may be a DNA probe that is complementary to the gene you want to clone; or it could be an antibody that recognizes the protein that is encoded by the gene. E6. A hybrid vector is a vector that has a piece of “foreign” DNA inserted into it. The foreign DNA came from somewhere else, like the chromosomal DNA of some organism. To construct a hybrid vector, the vector and source of foreign DNA are digested with the same restriction enzyme. The complementary ends of the fragments are allowed to hydrogen bond to each other (i.e., sticky ends are allowed to bind), and then DNA ligase is added to create covalent phosphoester bonds. If all goes well, a piece of the foreign DNA will become ligated to the vector, thereby creating a hybrid vector. As described in Figure 18.2, the insertion of foreign DNA can be detected using X-Gal. As seen here, the
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This note was uploaded on 04/07/2009 for the course BIO 325 taught by Professor Saxena during the Spring '08 term at University of Texas.

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E18 - E1 Sticky ends which are complementary in their DNA...

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