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Uses for PCR based technology
- Gene therapy
- genotyping
- Transgenics (GMO's)
- sequencing
- mapping
- cloning
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Recombinant DNA technology
- involves isolation & purification of genes
- uses genetic molecular markers (ie gfp or zeocin)
- makes modifications or reintroduces genes (ie lmp2a)
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3 things needed for Recombinant DNA
- 1. Restriction endonucleases
- 2. DNA ligase
- 3. cloning vectors
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Restriction cuts features
- Recognition sites are PALINDROMES (seq reads same as reverse)
- Sites are 4, 6, 8 bp in length
- Cleavages generate the following: blunt ends, 5' or 3' overhangs
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Steps in making & selecting for recombinant DNA
- 1. Cut foreign DNA - at Restriction enzyme site
- 2. Transform bacteria
- 3. Plate bacteria and see growth - if cut in between a tetR for example, will not be resistant to tet
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Blue-White selection
- Utilizes lac z - which makes B-Galactosidase
- X-Gal reacts with B-galactosidase & makes blue precipitate
- When you plate transformed back on X-gal, the cut thru lac z will be white
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Restriction Maps
- Restriction enzymes used to ORDER the recognition sites on piece of DNA
- (do examples as in HW III #1)
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