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Gene Technology

Recombinant DNA Techniques – Basic tools – part 1. Gene Technology. Recombinant (organism) – General term for creation of an organism by insertion of foreign genetic material (genes, promoter); -> cloning. Why do we want to clone genes?.

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Gene Technology

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  1. Recombinant DNA Techniques – Basic tools – part 1 Gene Technology Recombinant (organism) – General term for creation of an organism by insertion of foreign genetic material (genes, promoter); -> cloning Why do we want to clone genes? • Creating model organism -> study gene regulation (disease genes) -> Medicine • Production of recombinant protein (pharmaceutical products, food additives, laundry industry, …) -> Industry • Engineering of proteins -> Industry

  2. Gene Technology Methods and applications of genetic technology

  3. Gene Technology How do clone a gene? Clones -> Cells or organisms with identical DNA

  4. Gene Technology

  5. Gene Technology

  6. Gene Technology Restrictionendonucleases Type II 5’-> 3’ 3’ <- 5’

  7. Gene Technology Restriction endonucleases -> cut ds DNA (not ss DNA)

  8. Gene Technology Electrophoresis DNA interacts with EtBr -> illuminated with UV light -> fluorescence signal

  9. Gene Technology X-Ray structure of a complex of ethidium bromid with DNA. Gel Electrophoresis Agarose gels: 0.5 % - 2.5 % in 1 x TAE or 0.5 x TBE Acrylamid gels: 6 % - 12 % in 1 x TBE Agarose gel Polyacrylamid gel Separation on agarose gel: 500bp – 20 kbp Separation on acrylamid gel: 1- 2000 bp

  10. Gene Technology Electrophoresis – Restriction analysis (maps)

  11. Gene Technology Construction of a restriction map. Restriction map for the 5243-bp circular DNA of SV40.

  12. Gene Technology Creation of Recombinant Plasmid

  13. Gene Technology Ligases -> form phosphodiester bonds

  14. Gene Technology Plasmid cloning vector

  15. Gene Technology Plasmid cloning vector

  16. Gene Technology Viral cloning vector -> Bacteriophages Bacteriophage Lamda (λ): Genes for lysogenic cycle Genes for lytic cycle -> lysogenic cycle not really essential for this process -> genes can be replaced -> around 15 kb

  17. Gene Technology Plaques – lyst cells apon infection with bacteriophages

  18. Gene Technology Vector systems for Cloning

  19. Gene Technology The 5 basic steps of cloning If cut DNA coming from 1 fragment -> just colonies with no insert + colonies with the same insert -> normal cloning step !!! If cut DNA coming from different fragments (whole genome of organism) -> colonies with no insert + Colonies with different inserts -> library !!! Within one colony all cells have the same insert -> clones !!!

  20. Gene Technology Development of a cloning strategy

  21. Gene Technology Cloning Experiment Important features for a successful cloning experiment: -> Creation of the recombinant plasmid -> Transformation -> Selection

  22. Gene Technology Gene transfer into the host organism -> depended on type of cells • In Prokaryotes: • - Transformation -> uptake of naked DNA (chemical transformation, electroporation) • - Conjugation -> DNA transfer by cell – cell contact • Transduction -> DNA transfer by bacteriopage infection • In Eukaryotes:->used with fungi, animal and plant cells: • Electroporation • Microinjection • protoplasts

  23. Gene Technology Choosing the right start material: -> Type of DNA -> dependent of origin of DNA and purpose of cloning 1. Chromosomal DNA -> genes and regulatory elements of prokaryotes -> regulatory elements of eukaryotes 2. cDNA (complementary DNA) -> genes of eukaryotes 3. Vector DNA -> dependend on insert size -> Purity of DNA or RNA -> important for successful cloning (proteins or RNA impurity) -> impurity (RNA or DNA impurities) will also be present in library Insert

  24. Gene Technology Choosing the right start material -> Plasmid DNA isolation

  25. Gene Technology Choosing the right start material -> preparation of genomic DNA • Grow organism ( prokaryotic and eukaryotic) • Isolate genomic DNA • Fragmentation (partial digestion) of genomic DNA with restriction enzymes

  26. Gene Technology Choosing the right start material -> preparation of cDNA Eukaryotic cells use RNA Polymerase II for transcription of genes -> just RNA Polymerase II transcripts (mRNA) have a polyA tail !!! • Grow organism (eukaryotic) under conditions to induce production of protein (mRNA) of interest • Isolate mRNA

  27. Gene Technology Choosing the right start material -> preparation of cDNA • Grow organism (eukaryotic) under conditions to induce production of protein (mRNA) of interest • Isolate mRNA • Create cDNA based on mRNA

  28. Gene Technology Directional cloning of cDNA

  29. Gene Technology Gene Library DNA fragments part of the genome -> Library of one organism -> Used to screen for gene of interest Clones -> genetically identical

  30. Gene Technology Check for successful cloning -> Insertional inactivation • Gene in cloning site: • LacZ -> pUC18 (lacZ complements the host defect in lacZ) • -> pUC18 into host organism -> active lacZ (β-galactosidase) -> cleavage of X-gal (blue colonies) • -> gene cloned into polylinker -> lacZα gene disrupted -> no cleavage of X-gal (white colonies)

  31. Gene Technology Different types of libraries

  32. Gene Technology Different types of libraries • Genomic Library-> represents the complete sequence of an organism • -> in prokaryotes: used to clone genes, promoters, … • -> in eukaryotes: used to clone promoters, regulatory • elements • -> all fragments are almost equally represented in library • cDNA Library -> represents all genes expressed under these conditions • -> in eukaryotes: used to clone genes • -> all cDNAs are not equally -> depended on mRNA level of transcript

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