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Recombinant DNA Technology

Recombinant DNA Technology. r-DNA techniques. Isolation of Cellular DNA and RNA Plasmids Ability to disrupt cells and recover intact, pure DNA (or RNA) Reverse Transcriptase Restriction enzymes Sequence specific DNA cutting reactions Transformation techniques CaCl2 method Electroporation

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Recombinant DNA Technology

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  1. Recombinant DNA Technology

  2. r-DNA techniques • Isolation of Cellular DNA and RNA • Plasmids • Ability to disrupt cells and recover intact, pure DNA (or RNA) • Reverse Transcriptase • Restriction enzymes • Sequence specific DNA cutting reactions • Transformation techniques • CaCl2 method • Electroporation • Phage/virus transduction

  3. Plasmids Extrachromosomal and non-essential. Usually only 1 type of plasmid per cell at any given time. Many copies of each type

  4. Isolation Techniques • Cells can be disrupted (lysed) nonmechanically under mild conditions to release intact chromosome and plasmid DNA. • Purified plasmid DNA can be separated from chromosomal DNA by precipitation • Chromosomal and plasmid DNA can be cut into well defined fragments and separated by size with agarose gel electrophoresis • mRNA transcripts can be isolated from their polyA tails and RT can be used to produce a DNA fragment corresponding the gene

  5. Cell Disruption and DNA Isolation Treat cell with lysozyme (cuts G-M bond) Gentle shaking lyses cell and releases the DNA Add ethanol to precipitate chromosomal DNA and keep plasmid DNA in solution

  6. cDNA via Reverse Transcriptase Isolate mRNA thru poly A tail Use RT to create complimentary DNA strand. Use DNA polymerase to make second DNA strand

  7. Restriction Enzymes • Restriction enzymes were discovered which allow one to cut DNA in specific places • The fragments can be isolated by size (electrophoresis) • The different fragments can be spliced together in different ways to create “new genes” in a very directed manner. The new genes can be spliced into stable molecular “vectors”

  8. Restriction Sites hindIII,ecoR1,etc ATGCAT TACGTA Symmetrical

  9. Plasmid Structure Selection Protein DNA here Promoter

  10. Plasmid Organization

  11. Transformation • Various methods for adding DNA to living cells • CaCl2 method • Treat “competent” cells with cold CaCl2 solution. • Mix with DNA and select for transformed cells • Phage and virus transduction • Infect cells with phage or viruses containing desired genes • Electroporation • Establish strong electric field in cell suspension. Add desired DNA. Select for cells that have been transformed (reporter genes)

  12. Cold CaCl2 Method Add plasmid Transformed cells

  13. Transducing Phage Plasmid is injected by phage then integrates into chromosome

  14. Electroporation Add DNA Cell suspension

  15. Summary • r-DNA technology is made possible by 3 methods. • DNA isolation techniques esp. plasmids • Restriction enzymes and ligation techniques • Tranformation techniques

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