Genetics Block 3 Notes

Genetics Block 3 Notes - 19 Recombinant DNA Technology What...

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19) Recombinant DNA Technology What does it do: isolation of a single gene from a genome; virtually unlimited copies; produces combinations of DNA that do not occur naturally. Unbeknownst How is it done: involves the enzymatic clevage of DNA, joining the 2 fragments together to make a new DNA and finally introduce the new DNA into a living cell for replication. Why is it done: Isolation of specific gene; produce large quantities; Modification of existing organisms; correction of genetic defects. Problems occur in application to humans. Restriction endonucleases : Bacterial proteins that are capable of sequence-specific binding to double stranded DNA and cleaving both strands; Bacterial immune system, protecting bacteria against viral infection = restriction enzyme Over 250 sequence-specificities; first isolated was EcoRI (E Coli strain RY13) Restriction enzymes cut DNA at specific target sequences(palindromes ), the frequency of their clevage and therefore the size of the related fragments is dependent on the number of bases in the recognition sequence. 4 base pair sequences – 4 4 = 1 site every 256 base pairs Distance between enzyme target sites can be determined by running digest through agarose gel. Complemntary single-stranded ends (sticky ends ) join other molecules with the same enzyme. Vectors : carrier DNA molecules that transfer and replicate inserted DNA fragments in host organisms; host specific (mammalian-mammalian, bacterial-bacterial); limited by size; independently replicates itself and the inserted DNA fragments (origin of replication ); contain unique clevage sequences for restriction enzymes; selectable markers yield easy identification of cells that contain the vector. Plasmids – DNA circular molecules that encodes an origin of replication, an antibiotic resistance gene, and a synthetic region of multiple unique restriction enzyme clevage site; Taken up by transformation (as naked DNA); limited to smaller fragments (>10 kb). Lambda phage – replace phage genome with foreign DNA fragments; must be treated with phage proteins before it can infect bacterial cells; larger fragments (up to 20 kb). Artificial chromosomes – bacterial (BAC ) or yeast (YAC ). BAC are based on F factor, contain anitbiotic resistance gene, and can accomodate inserts up to 300 kb. YAC has telomeres at each end, an origin of replication, a centromere, selectable markers, and accomodate inserts up to 1000 kb. Cloning : Step 1: Digest the vector DNA and the foreign DNA to be cloned with the appropriate restriction enzyme. Ends of cleaved DNA may vary: sticky (overhang) or blunt (no overhang). The restriction enzyme digested vector DNA and insert DNA are combined and joined into a single recombinant DNA molecule by incubation with a DNA ligase enzyme. = making recombinant molecule; unnatural. Step 2: Host-cells take up and amplify the vector-insert recombinants; plasmid vectors are added
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This note was uploaded on 05/29/2008 for the course BIOLOGY 2203 taught by Professor Anthwal during the Spring '08 term at Temple.

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Genetics Block 3 Notes - 19 Recombinant DNA Technology What...

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