Recombinant DNA Technology

1. Recombinant DNA Technology Methods for Isolating, Amplifying and Studying Specific DNA Sequences

2. Cutting and Joining DNA Fragments Restriction Enzyme: cleaves DNA at specific sequences DNA Ligase: joins DNA fragments by sealing nicks in backbone

3. sticky end Results in sticky end Use of the Restriction Enzyme Bam H1 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ 5’— G G A T C C — 3’ 3’— C C T A G G — 5’ DNA fragments with complementary sticky ends can associate to form new DNA combinations.

4. Viewing DNA Fragments: Agarose Gel Electrophoresis

5. Locating DNA Fragments by Southern Blotting Probe is complementary to DNA of interest DNA Fragments are transferred to membrane A subset of fragments binds to the probe

6. Cloning Genes • Gene cloning = isolation of a specific segment of DNA in a form that can be duplicated in another cell • Requires a • Vector = carrier of DNA segment • Host = cell in which foreign DNA can be duplicated and its gene product may be synthesized • Recombinant DNA = DNA produced by joining fragments from two or more different sources

7. ampR Bam H1 tetR ampR tetR gene disrupted, no longer functional Cloning Vectors • For Bacterial Hosts • Plasmid

8. Cloning Vectors • For Bacterial Hosts • Bacteriophage • Cosmid • Expression Vector

9. Cloning Vectors • For Eukaryotic Hosts • Shuttle Vector • YAC = yeast artificial chromosome • Ti plasmid

10. Tools for Finding Genes • DNA library = collection of clones from one DNA donor • Categorized by: vector, source of DNA • Genomic library = all of the sequences from the genome of a single organism • cDNA library= complementary DNA, made using mRNA as a template

11. Producing a Genomic Library Source of DNA for Library Many recombinant molecules are formed, each with a different DNA insert

12. Producing cDNA Primer binds to poly-A tail DNA produced from RNA template Partial digestion of RNA template Second DNA strand produced using remaining RNA segments as primers Gaps in backbone sealed

13. AATGCCAT Denature DNA by heating TTACGGTA AATGCCAT TTACGGTA Tools for Finding Genes Probe: sequence of DNA or RNA that is complementary to the gene of interest; Used to locate a copy of the gene in a library by hybridization TTACGGTA Add Probe Probe Binds to gene

14. Screening a Library with a Probe

15. Amplifying DNA with the Polymerase Chain Reaction (PCR) • Copies a specific region in the DNA • Can be used with small amounts of sample • Uses repeated cycles of heating to denature DNA and cooling to synthesize new DNA • Requires • primers to begin synthesis • heat-stable polymerase (Taq polymerase)

16. Polymerase Chain Reaction

17. H H OH H deoxyribose dideoxyribose DNA Sequencing by the Sanger Method • Relies on Chain Termination during replication due to incorporation of Dideoxynucleotides

18. Unknown Sequence 3’-A-G-C-C-C-A-G-A-T-T-5’ • Replicate unknown DNA using dideoxynucleotides to terminate growing chains at various positions 5’-T-C-G-3’ Complementary Products Black= dideoxynucleotide 5’-T-C-G-G-G-T-C-T-3’ 5’-T-3’ 5’-T-C-3’ 5’-T-C-G-G-G-3’ 5’-T-C-G-G-G-T-C-T-A-3’ 5’-T-C-G-G-G-T-C-T-A-A-3’ 5’-T-C-G-G-G-T-C-3’ 5’-T-C-G-G-G-T-3’ 5’-T-C-G-G-3’

19. Dideoxynucleotide A T C G • Use gel electrophoresis to separate replicated fragments that differ by one base • Size of DNA on gel shows distance from start of replication • Labeling of dideoxynucleotide shows complementary sequence

20. Dideoxynucleotide A T C G Complementary Sequence Unknown Sequence • Determine unknown sequence from complementary sequence with base-pairing rules Complementary Sequence 5’-T-C-G-G-G-T-C-T-A-A 5’-T-C-G-G-G-T-C-T-A 5’-T-C-G-G-G-T-C-T 5’-T-C-G-G-G-T-C 5’-T-C-G-G-G-T 5’-T-C-G-G-G 5’-T-C-G-G 5’-T-C-G 5’-T-C 5’-T

23. Applications of Recombinant DNA Technology Genetic Testing and Gene Therapies Products of Biotechnology

24. RFLP Analysis • RFLP = Restriction Fragment Length Polymorphism • Identifies differences in the length of restriction fragments derived from similar DNA sequences • Analyzed by Southern Blotting • Used in gene mapping

25. RFLP Analysis in Paternity Testing

26. RFLP Analysis in Forensics 1 2 3 CSRCIE MNEE 4 5 6 7 Suspects Suspects

27. DNA Fingerprinting • Detects patterns for short repeated sequences that are highly variable among humans • Described as • VNTR: Variable Number of Tandem Repeats • STR: Short Tandem Repeats • Microsatellites Analysis using Microsatellite sequences

28. Genetic Testing

29. Gene Therapy • Andrew Gobea was born with SCID, an immune deficiency due to a defect in the ADA gene. • Four days after his birth, he received an injection of his own stem cells carrying functional ADA genes. • Andrew’s immune system began producing ADA, but the amount was not sufficient to maintain full health. He needs daily injections of ADA enzyme.

30. siRNA for ApoB was delivered in lipid capsules RNA Interference as a Possible Treatment for High Cholesterol • High levels of ApoB, a protein component of blood lipid carriers, are associated with high cholesterol levels • In monkeys, siRNA for ApoB has lowered the mRNA for ApoB in liver cells and also reduced serum cholesterol levels

31. Recombinant DNA Products

32. Genetically Modified Organisms • Introducing or modifying specific genes to alter the phenotype of an organism • Transgenic: organism that contains a gene from another species in all of its cells • Transgenic Animals • Models of Human Disease • Produce Pharmaceuticals

33. Studying Gene Function with Knockout Mice • neo+ gene gives resistance to G418 • tk+ gene gives sensitivity to gancyclovir • Insertion of neo+ into target gene yields neo+ and tk- cells, resistant to G418 and to gancyclovir

34. Round Up Ready Soybeans are resistant to herbicide “Golden” rice with beta-carotene and extra iron Bt Corn produces its own pesticide Transgenic Plants

35. What Are Some Concerns?