DNA Technology and Genetic Engineering

1. Human protein production Transgenic organisms Forensic analysis Human genome project RFLP analysis DNA Technology and Genetic Engineering

2. DNA Technology and Genetic Engineering (making changes in DNA) Nucleus Chromosome • 1: Production of human proteins • 2: To identify people • 3: To identify human diseases • 4: To identify all human genes • 5: To genetically engineer food Cell Coils Supercoils Nucleosomes Histones DNA

3. Use 1: To produce human proteins in bacteria (E. coli) Example: Curing Pituitary Dwarfism

4. What is Dwarfism? • Dwarfism is a recessive disease that causes adults to be no more than 4 feet tall • Little people produce little or no growth hormone, which is made by the pituitary • Researchers studied families with dwarfism and found that people with dwarfism have defective copies of the gene, GH1

5. Early attempts to treat Dwarfism • Attempts to inject growth hormone from pigs (a strategy that worked previously for insulin) did not work – only GH from humans would work (until 1982 source from human cadavers and up to 20,000 pituitaries were needed!) • Some of these pituitaries were contaminated with prions, which cause degenerative brain disorders

6. Solution: Use recombinant DNA to produce GH in bacteria! • Recombinant DNA = DNA that results from combining DNA from different sources • ex. mouse + human DNA • human + bacterial DNA

7. Recombinant DNA Overview Cell containing geneof interest 1 Bacterium Plasmidisolated 2 DNA isolated 3 Gene inserted into plasmid Bacterialchromosome Plasmid Gene ofinterest Recombinant DNA(plasmid) DNA 4 Plasmid put intobacterial cell Recombinantbacterium 5 Cell multiplies withgene of interest Copies of gene Copies of protein Gene for pestresistanceinserted intoplants Clones of cell Protein used to make snow format highertemperature Gene used to alter bacteriafor cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy

8. How Do You Make Recombinant DNA? • How do you make recombinant DNA? We need • A) To isolate genes with restriction enzymes • B) A vector • C) To combine the genes and the vector • What do we do with it? • D) Transfer the recombinant DNA to the host • E) Find the gene of interest (human growth hormone)

9. A) Isolate genes with restriction enzymes (DNA scissors) • Occur naturally in bacteria – why? • Bacteriophages infect bacteria • Cut up foreign DNA • Hundreds are purified and available commercially • Recognize and cut at specific base sequences in DNA (usually 4-8 bases long)

10. Products generated by restriction enzymes • A) Sticky-end cutters EnzymeRecognition siteDNA after cuts • B) Blunt-end cutters EnzymeRecognition siteDNA after cuts 5’...GAATTC...3’ 3’...CTTAAG...5’ 5’...G 3’...CTTAA AATTC...3’ G...5’ EcoRI  5’...CCCGGG...3’ 3’...GGGCCC...5’ 5’...CCC 3’...GGG GGG...3’ CCC...5’ SmaI 

11. A) Isolate genes with restriction enzymes (DNA scissors) • Take genomic DNA (in this case, human cells) and cut it with a particular restriction enzyme • MANY restriction fragments formed • ALL parts of the DNA are cut whenever there is a restriction site • Now what? Put these fragments in a vector

12. B) Vectors • Vector = something to carry the gene of interest into the host (i.e. bacteria) • A. Mechanical – micropipettes or gene guns • B. Biological – virus or plasmid • Plasmid – additional, free-floating ring of DNA found only in bacteria • Can replicate within a cell (has origin of replication) • Has antibiotic resistance gene • Cut both the plasmid and gene with the same restriction enzyme and their ends will hydrogen bond = gene splicing

13. Restriction enzymerecognition sequence 1 DNA Restriction enzymecuts the DNA intofragments Restriction enzymecuts the DNA intofragments 2 C) Combine the gene of interest and the vector by sealing ends with DNA ligase… now you have made recombinant DNA Sticky end Addition of a DNAfragment fromanother source 3 Two (or more)fragments sticktogether bybase-pairing 4 DNA ligasepastes the strand 5 Recombinant DNA molecule

14. C) Combine the gene of interest and the vector – a differentpicture

15. D) Transfer the Recombinant DNA to the Host (Transformation) • Recombinant DNA is transferred to a host cell. • Can use heat-shocking or electricity to get plasmid into bacteria • How do we know which bacterial cells have our plasmid? • Antibiotic resistance gene! • Resistance gene allows ONLY those bacteria with the plasmid to grow in media that have an antibiotic on it • When the host cell copies its DNA (replicates), it also makes a copy of the plasmid. Results in clones, which are genetically identical copies.

16. D) Transfer the Recombinant DNA to the Host • We use bacteria because they reproduce very quickly and have all the protein synthesis machinery (enzymes, ribosomes) • Insulin for Type I diabetics • Blood factor VIII-hemophilia (clotting factor) • Antigens for vaccines (Hep B, flu, meningitis) • Cutting chromosomes in order to study individual pieces

17. Host Cells Produce Protein Products – ex. GH and insulin

18. E) Find the gene of interest • Each clone consists of identical cells containing one fragment (of many) of human DNA • The collection of all the cloned DNA fragments is known as a genomic library • Each fragment represents a “book” that is “shelved” in plasmids inside bacterial cells • Thus, it is a library of all the organism’s genes Genome cut up with restriction enzyme Recombinantplasmid Recombinantphage DNA OR Phage clone Bacterialclone Plasmid library Phage library Figure 12.6

19. E) *Side note – libraries can also be made from cDNA • The enzyme reverse transcriptase can be used to make a smaller library, called a cDNA library • This contains only the genes that are expressed (transcribed) by a specific cell type (rather than ALL the genes found in an organism) • These genes can then be digested and placed in vectors • Why is this useful? • Bacterial mRNA does not have introns – doesn’t have the machinery to splice eukaryotic genes • Can help a researcher study the genes responsible for specialized functions of a certain cell type

20. CELL NUCLEUS Exon Intron Exon Intron Exon DNA ofeukaryoticgene Transcription 1 RNA transcript RNA splicing(removes introns) 2 mRNA Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand 3 TEST TUBE Reverse transcriptase Breakdown of RNA 4 cDNA strand Synthesis of secondDNA strand 5 cDNA of gene(no introns)

21. E) Find the gene of interest • How do we find the right “shelf” in the library? • If we know at least part of the DNA sequence, we can create a nucleic acid probe • Probe = radioactively labeled single-stranded DNA piece that can base pair with a particular sequence • Ex: ATCCGA • The probe is mixed with clones that have been heated or treated with a chemical to separate the DNA strands • The probe will “tag” the correct “shelf” or bacterial clone that contains the gene of interest

22. E) Find the gene of interest Radioactiveprobe (DNA) Mix with single-stranded DNA fromvarious bacterial(or phage) clones Single-strandedDNA Base pairingindicates thegene of interest

23. Bacterial colonies containingcloned segments of foreign DNA E) Find the gene of interest Radioactive DNA Transfercells tofilter 1 Solutioncontainingprobe Filterpaper The bacterial colony can then be grown and the protein of interest can be isolated in large amounts Treat cellson filter toseparateDNA strands Add probeto filter ProbeDNA 2 3 Gene ofinterest Single-strandedDNA from cell Hydrogen-bonding Autoradiography 4 Colonies of livingcells containinggene of interest Developed film Compare autoradiographwith master plate 5 Master plate

24. 1 Isolate DNAfrom two sources Human cell E. coli E) Isolating the gene of interest 2 Cut both DNAs with the same restriction enzyme Plasmid DNA Gene V Sticky ends 3 Mix the DNAs; they joinby base-pairing 4 Add DNA ligaseto bond the DNA covalently Recombinant DNAplasmid Gene V 5 Put plasmid into bacteriumby transformation 6 Clone the bacterium Bacterial clone carrying manycopies of the human gene

25. Mass-Produced Genes