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Tools of Genetic Engineering: Understanding DNA Manipulation Techniques

This unit explores essential tools utilized in genetic engineering, which involves the manipulation of DNA and genes in organisms. We delve into various methods including cutting DNA with restriction enzymes, pasting using ligases, and copying DNA through techniques such as PCR and plasmids. We highlight the work of pioneering scientists and the evolution of these methods since the discovery of restriction enzymes in the 1960s. By examining these foundational tools, you will gain a comprehensive understanding of the mechanics behind DNA transformation and recombinant DNA technology.

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Tools of Genetic Engineering: Understanding DNA Manipulation Techniques

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  1. Biotechnology today • Genetic Engineering • manipulation of DNA • if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with • this unit is a survey of those tools… Our tool kit…

  2. Cut, Paste, Copy, Find… • Word processing metaphor… • cut • restriction enzymes • paste • ligase • copy • plasmids • bacteria • transformation • PCR • find • Southern blotting / probes

  3. Cut DNA • Restriction enzymes • restriction endonucleases • discovered in 1960s • evolved in bacteria to cut up foreign DNA (“restriction”) • protection against viruses & other bacteria • bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA

  4. What do you notice about these phrases? radar racecar Madam I’m Adam Able was I ere I saw Elba a man, a plan, a canal, Panama Was it a bar or a bat I saw?

  5. Madam I’m Adam Restriction enzymes • Action of enzyme • cut DNA at specific sequences • restriction site • symmetrical “palindrome” • produces protruding ends • sticky ends • Many different enzymes • named after organism they are found in • EcoRI, HindIII, BamHI, SmaI CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC 

  6. 1960s|1978 Discovery of restriction enzymes Werner Arber Daniel Nathans Hamilton O. Smith Restriction enzymes are named for the organism they come from: EcoRI = 1st restriction enzyme found in E. coli Restriction enzyme movie

  7. GAATTC GAATTC CTTAAG CTTAAG Biotech use of restriction enzymes DNA Restriction enzyme cuts the DNA Sticky ends (complementary single-stranded DNA tails) AATTC AATTC G G G CTTAA G CTTAA Add DNA from another source cut with same restriction enzyme AATTC G G AATTC CTTAA G DNA ligase joins the strands. GAATTC Recombinant DNA molecule CTTAAG

  8. Paste DNA • Sticky ends allow: • H bonds between complementary bases to anneal • Ligase • enzyme “seals” strands • bonds sugar-phosphate bonds • covalent bond of DNA backbone

  9. Copy DNA • Plasmids • small, self-replicating circular DNA molecules • insert DNA sequence into plasmid • vector = “vehicle” into organism • Original plasmid is call a cloning vector • transformation • insert recombinant plasmid into bacteria • bacteria make lots of copies of plasmid • grow recombinant bacteria on agar plate • clone of cells = lots of bacteria • production of many copies of inserted gene DNA RNA  protein  trait

  10. Intron problem • Gene sequence must be made without introns? • Take mRNA after introns are cut out and use reverse transcriptase to make copy DNA which is DNA without intron sequences. • Bacteria can’t process introns later.

  11. Recombinant plasmid • Antibiotic resistance genes as a selectable marker • Restriction sites for splicing in gene of interest • Selectable marker • Plasmid has both “added” gene & antibiotic resistance gene • If bacteria don’t pick up plasmid then die on antibiotic plates • If bacteria pick up plasmid then survive on antibiotic plates • selecting for successful transformation selection

  12. Selection for plasmid uptake • Ampicillin becomes a selecting agent • only bacteria with the plasmid will grow on amp plate only transformed bacteria grow all bacteria grow LB plate LB/amp plate

  13. restriction sites insertedgeneof interest LacZ gene brokenLacZ gene X lactose  white color lactose  blue color recombinantplasmid plasmid amp resistance amp resistance origin ofreplication Need to screen… • Need to make sure bacteria have recombinant plasmid EcoRI all in LacZ gene BamHI HindIII

  14. LacZ is a screening system • Make sure inserted plasmid is recombinant plasmid • LacZ gene on plasmid produces digestive enzyme • lactose(X-gal)  blue • blue colonies • insert foreign DNA into LacZ gene breaks gene • lactose (X-gal)  blue • white colonies • white bacterial colonies have recombinantplasmid X X We wantthese!!

  15. Amp selection & LacZ screening • gene of interest • LacZ gene • - amp resistance LB/amp LB/amp/Xgal

  16. Recombinant DNA movie Gene cloning

  17. Cut, Paste, Copy, Find… • Word processing metaphor… • cut • restriction enzymes • paste • ligase • copy • plasmids • bacteria • transformation • PCR • find • Southern blotting / probes    

  18. Any Questions??

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