1. Recombinant DNA Technology restriction enzymes making recombinant DNA molecules expression vectors applications of recombinant DNA technology Forensic DNA Profiling short tandem repeats DNA fingerprints References: p. 514-517; 519-523; 525-526; 543-544

2. Bacterial restriction endonucleases A restriction endonuclease is an enzyme that recognizes a specific nucleotide sequence in DNA (a restriction site) and makes a cut at that site. restriction sites are palindrome sequences restriction digestion of DNA results in molecules with complementary single-strand tails (sticky ends)

3. Restriction endonucleases

4. Creating recombinant DNA molecules Putting together pieces of DNA form different origins

5. Use of the pUC19 expression vector amp R is the ampicillin resistance gene the polylinker region inside the lacZ + gene is a cluster of restriction sites; any of which maybe chosen to insert a gene of interest (and this would disrupt the lacZ + gene) any gene inserted in the polylinker region will be under the control of the T7 virus promoter (which is recognized by the T7 RNA polymerase) and the lac operator (lacO, the binding site of the lac operon repressor, which is coded by lacI) the host bacterium for this plasmid is a cell that has been engineered to carry the viral T7 RNA polymerase gene under the control of the lac operon promoter and operator in its genome

6.  IPTG is a lactose analog and can therefore induce the expression of both the T7 RNA polymerase gene in the host genome and of any gene in the plasmid polylinker (by binding the lac operon repressor and inactivating it). Use of the pUC19 expression vector

7. Cloning vector pUC19

8. Creating recombinant DNA molecules with a cloning vector

9. The use of antibiotics for selection Antibiotic selection: if the nutrient growth medium also contains an antibiotic, the only bacteria that will be able to multiply and form colonies will be those that carry the correct antibiotic resistance gene. Example: amp R in pUC19.

10. The pUC18 polylinker region inside the lacZ + gene pUC18 is very similar to pUC19.

12. The Xgal blue-white assay to identify E. coli colonies that contain cloned DNA fragments To screen for cells that have picked up a plasmid with a cloned DNA fragment following transfection, cells are grown on agar medium containing X-gal, the antibiotic ampicillin, and IPTG. any bacterium that takes up a plasmid will become resistant to ampicillin and will grow to form a colony recombinant plasmids (those with a foreign DNA fragment inserted in their polylinker regions) will have a disrupted lacZ + gene and will not express  -galactosidase plasmid vectors that did not integrate a foreign DNA fragment in their polylinker regions will have an intact lacZ + gene and will express  -galactosidase

13. The Xgal blue-white assay to identify E. coli colonies that contain cloned DNA fragments The enzyme  -galactosidase cleaves X-gal into galactose and 5-bromo-4-chloro-3- hydroxyindole, which is then oxidized to an insoluble blue dye.

15. The Xgal blue-white assay to identify E. coli colonies that contain cloned DNA fragments Blue colonies (X-gal positive) carry plasmid vectors without cloned foreign DNA. White colonies (no Xgal reaction) carry plasmids with cloned foreign DNA.

16. The Agrobacterium Ti plasmid can be used to transfer genes into plants TL and TR: flanking sequences that are required for the transfer of the DNA segment from bacteria to the plant cell.

17. The Agrobacterium Ti plasmid can be used to transfer genes into plants

18. Generating transgenic crops with glyphosate resistance Glyphosate is a herbicide that kills plants by inhibiting EPSP synthase, a chloroplast enzyme that is essential for amino acid biosynthesis. EPSP synthase can be expressed in larger than normal quantities by plants that carry the EPSP gene under the control of the cauliflower mosaic virus (CMV) promoter in a Ti plasmid vector plant crops that carry this integrated plasmid are resistant to glyphosate concentrations that would kill normal plants

19. (a strong promoter)

20. Callus: a cluster of undifferentiated cells that results from the proliferation of a Ti-transformed cell in culture. A plant cell callus can regenerate a whole plant.

21. Gene targeting in mouse ES cells Mutant organisms can be created to study the effect of mutant alleles in vivo. Knockout mice can be obtained after homologous recombination of a cloned mutant allele with the cellular gene in embryonic stem (ES) cells. Generation of knockout ES cells: the cloned gene of interest (target) is disrupted with a segment of DNA containing the neomycin resistance gene (neo + ) the disrupted gene is placed in a vector that also contains the thymidine kinase (tk + ) gene from the herpes simplex virus (thymidine kinase can phosphorylate the thymidine analog glanciclovir, turning it into a toxic inhibitor of DNA replication and killing the cell) the vector is introduced into mouse ES cells

22. Gene targeting in mouse ES cells Two thing may happen in the ES cells: the homologous recombination of the disrupted target gene with the cellular gene (knockout), replacing a copy of the normal cellular target with the disrupted version (these cells will be able to grow in medium containing both neomycin and ganciclovir because the tk + gene will not be integrated) the random integration of the entire vector into the cellular genome without knocking out the normal cellular target (these cells will not be able to grow in medium containing ganciclovir because the tk + gene will be integrated and expressed, resulting in toxicity)

23. Creating knockout mice

24. Once created, the knockout ES cells are injected into a mouse embryos. knockout ES cells from a black mouse are injected into the embryo from a white mouse the hybrid embryo is implanted in a foster mother and develops into a chimeric mouse (which may have black mouse germ cells) the chimera mice are mated to obtain homozygous knockout mice (black)

26. Gene therapy Components of the Moloney Murine Leukemia Virus (MLV) genome: the  gene, necessary for encapsulation structural genes of the virus - gag: coat proteins - pol: reverse transcriptase - env: envelope glycoproteins 5’ and 3’ long terminal repeats (LTRs) In the SAX virus, all the structural genes of MLV have been replaced: neo r : neomycin (antibiotic) resistance SV40: simian virus promoter/enhancer hADA: Human adenosine deaminase (ADA) gene

27. Gene therapy The SAX virus can be encapsulated but will not reproduce. It can be used as a gene therapy vector to correct severe combined immunodeficiency (SCID), a hereditary disease caused by a defect in the hADA gene.

28. Gene therapy

29. Examination of the human genome

30. Forensic DNA profiling Forensic DNA profiling is the use of genetic markers to distinguish individuals and answer questions of interest to the legal system. the markers used are polymorphic loci that contain multiple repeats of a small unit (a repetitive sequence) the number of repeats of the unit sequence in each polymorphic marker locus vary widely among individuals, and their electrophoretic profile yields DNA fingerprints that are unique for each individual

31. Short tandem repeats STRs are clusters of tandem repeats of 2-9 nucleotide sequences. the lengths of the clusters depend on the number of sequence repeats in each allele because the small sequence sizes, DNA analysis can be done by using a method that is quick and requires very little tissue sample: the Polymerase Chain Reaction (PCR) the results yield a DNA fingerprint STR loci are also known as PCR loci (STRs)

32. 1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt 61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa 121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat 181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg 241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc 301 cctctgagtt tttgatacct cagattttaa ggcc Short tandem repeats D7S280 is one of the 13 core CODIS STR genetic loci. This DNA is found on human chromosome 7. In this particular example, the chromosome has 15 repeats of the GATA sequence. The number of repeats may be different in the other chromosome or in another individual. The D7S280 STR locus

33. The FBI’s 13 core STR loci

35. The amelogenin test Amelogenin is a protein found in the developing tooth enamel. the gene (AMEL) is present in both the X and the Y chromosomes, but the X-linked version is shorter it can be used for sex determination of human DNA samples of unknown origin through PCR of intron 1 of the gene: - PCR of AMEL-X gives rise to a 106 bp product - PCR of AMEL-Y gives rise to a 112 bp product

36. DNA profile using several STRs DNA fingerprints from the victim, an tissue sample of unknown origin found at the crime scene, and three suspects:

37. Identification of September 11, 2001 victims: there were 2603 victims in the attacks to the World Trade Center, but only 293 whole bodies were recovered ~20,000 pieces of bone and tissue were found among the 1.6 million tons of debris (some pieces as small as a fingertip) laboratories collected personal items from the victims’ homes (~7000 razor blades, combs, toothbrushes, etc.) the method of choice for DNA analysis was STR Examples of DNA evidence in forensic identification

38. The people’s STR evidence in the 1995 O. J. Simpson murder trial: 7 PCR loci in O. J. Simpson’s blood, found at the foyer of Nicole Brown’s apartment and leading out of the crime scene to Bundy Drive 7 PCR loci in Nicole Brown’s blood, found in a pair of socks in Simpson’s bedroom 2 PCR loci in Ronald Goldman’s blood, found on a pair of bloody gloves at Simpson’s home