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Chapter 10 Recombinant DNA Techniques 10.1 10.4 10.5 cloning DNA- basics transgenic organisms - reverse genetics genetic engineering © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) cut DNA with restriction enzyme take fragments reassemble in new combinations put back into organism (cell) transgenic organism 10.1 Recombinant DNA Techniques (gene cloning) restriction enzymes cut DNA at specific sequences restriction sites (palindromes) 10.1 Recombinant DNA Techniques (gene cloning) restriction enzymes sticky ends 5’ overhang 3’ overhang (complementary) blunt ends Fig. 10.2. Two types of cuts made by restriction enzymes © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) EcoRI restriction enzymes 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ 3’ 5’-----GAATT 3’-----C 5’ 5’ C-----3’ TTAAG-----5’ 3’ 10.1 Recombinant DNA Techniques (gene cloning) restriction enzymes EcoRI 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ 33’5’ 5’ 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ 5’3’ DNA ligase Fig. 10.1. Circularization of DNA fragments produced by a restriction enzyme © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) restriction enzymes vectors DNA sequence used to carry other DNA 10.1 Recombinant DNA Techniques (gene cloning) vectors •can be put in a host easily •contains a replication origin •have a gene for screening (eg. antibiotic resistance) 10.1 Recombinant DNA Techniques (gene cloning) vectors •for E. coli - plasmids l bacteriophage M13 Fig. 10.5. Common cloning vectors for use with E. coli © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) vectors put into cells via transformation electroporation Fig. 10.7. Construction of recombinant DNA plasmids containing fragments derived from a donor organism © 2006 Jones and Bartlett Publishers Fig. 10.4. Example of cloning © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) DNA to insert ? libraries collections of vectors (lots) each containing cloned DNA genomic cDNA 10.1 Recombinant DNA Techniques (gene cloning) genomic library (1) l phage cut with restriction enzyme x 10? “sticky ends” 10.1 Recombinant DNA Techniques (gene cloning) genomic library (2) cut with same restriction enzyme “sticky ends” 10.1 Recombinant DNA Techniques (gene cloning) genomic library (3) don’t forget DNA ligase …lots of different vectors 10.1 Recombinant DNA Techniques (gene cloning) cDNA library eukaryotic DNA has lots of introns genes are very large if we are only interested in the part of the gene that codes for protein… 10.1 Recombinant DNA Techniques (gene cloning) cDNA library (1) isolate the mRNA from the cell(s) oligo-dT column 10.1 Recombinant DNA Techniques (gene cloning) cDNA library (2) 5’-----------------AAAAAA-3’ mRNA use reverse transcriptase 3’-----------------TTTTTTT-5’ DNA 5’ -----------------AAAAAA-3’ DNA complementary DNA - cDNA then DNA polymerase… …a double stranded DNA from each mRNA 10.1 Recombinant DNA Techniques (gene cloning) cDNA library (3) ligate DNAs into vectors Fig. 10.8. Reverse transcriptase produces a single-stranded DNA complementary in sequence to a template RNA © 2006 Jones and Bartlett Publishers 10.1 Recombinant DNA Techniques (gene cloning) transformation or electroporation mix vectors (with insert) with cells libraries collections of vectors with different DNA inserts genomic cDNA great for abundant mRNA’s libraries mRNA in low copy number? RT-PCR reverse transcriptase-PCR What do you need to know to do PCR? More about plasmids nice to have lots of different single-site RE sites have to cut them open to put in insert (directional cloning) Fig. 10.9. (A) Diagram of the cloning vector pBluescript II (B) Sequence of the multiple cloning site showing the unique restriction sites [Data courtesy of Stratagene Cloning Systems, La Jolla, CA] © 2006 Jones and Bartlett Publishers More about plasmids (directional cloning) …G AATTCGATATCA AATTC-our - DNA-AAGCTT… …CTTAA GCTATAGTTCGA G-our - DNA-TTCGA A… EcoRI HindIII AATTC-our - DNA-A G-our - DNA-TTCGA More about plasmids need to have lots of different single site RE sites need to screen for bacteria that with the plasmid you only want to grow the bacteria took up the plasmid Fig. 10.9. (A) Diagram of the cloning vector pBluescript II (B) Sequence of the multiple cloning site showing the unique restriction sites [Data courtesy of Stratagene Cloning Systems, La Jolla, CA] © 2006 Jones and Bartlett Publishers More about plasmids need to have lots of different single site RE sites need to screen for bacteria that with the plasmid need to screen for plasmids with an insert some will have closed up without insert Fig. 10.10A,B. Detection of recombinant plasmids through insertional inactivation of a fragment of the lacZ gene from E. coli © 2006 Jones and Bartlett Publishers grow on ampicillin with Xgal Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] © 2006 Jones and Bartlett Publishers plasmid only plasmid with insert Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] © 2006 Jones and Bartlett Publishers Screening the library 106 to 10? of different clones How do you “find” the one you want ? Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Fig. 10.11. Colony hybridization © 2006 Jones and Bartlett Publishers Chapter 10 Recombinant DNA Techniques 10.1 10.4 10.5 cloning DNA- basics transgenic organisms - reverse genetics genetic engineering © 2006 Jones and Bartlett Publishers 10.4 Reverse genetics In the past… find mutant phenotype find mutant gene study wild-type gene Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4 Reverse genetics but now we can… mutate a gene find study the phenotype Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4 Reverse genetics transforming the germ line Drosophila P elements C. elegans mouse ESC domestic animals Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4 Reverse genetics transposase enzyme that can insert DNA flanked by inverted repeats can place itself randomly into the chromosome Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] •remove some of the inverted repeats -cannot be inserted and •insert DNA into coding region Fig. 10.18. Transformation in Drosophila mediated by the transposable element P © 2006 Jones and Bartlett Publishers your DNA + marker (eye color) Fig. 10.18. Transformation in Drosophila mediated by the transposable element P © 2006 Jones and Bartlett Publishers 10.4 Reverse genetics mouse put DNA into fertilized egg using engineered retrovirus Embryonic stem cells insert modified cells into blastocyst Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Fig. 10.19. Transformation of the germ line in the mouse using embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.] © 2006 Jones and Bartlett Publishers 10.4 Reverse genetics gene targeting fig. 10.20 Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Fig. 10.20. Gene targeting in embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.] © 2006 Jones and Bartlett Publishers 10.4 Reverse genetics Ti plasmid used on plants Agrobactgerium fig. 10.21 Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Fig. 10.21. Transformation of a plant genome by T DNA from the Ti plasmid © 2006 Jones and Bartlett Publishers 10.4 Reverse genetics Transformational rescue by using inserts of different lengths you can find out how much of the DNA is necessary Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] fig. 10.22 Fig. 10.22. Genetic organization of the Drosophila gene white © 2006 Jones and Bartlett Publishers Fig. 10.23. Eyes of a wildtype red-eyed male D. melanogaster and a mutant white-eyed male. [Courtesy of E. Lozovsky] © 2006 Jones and Bartlett Publishers 10.5 Genetic engineering applied Animal growth rate metallothionen promoter (very active) growth hormone Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] http://www.nytimes.com/2007/07/30/washington/30animal.html?_r=1&oref=slogin Even though these Atlantic salmon are roughly the same age, the big one was genetically engineered to grow at twice the rate of normal salmon. 10.5 Genetic engineering applied plants increase nutritional value b-carotene precursor to vitamin A in yellow vegetables high rice diets of lack b-carotene Fig. 10.25 Rice engineered to produce b-carotene 10.5 Genetic engineering applied plants rice with: b-carotene Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied plants rice also contains phytate which can causes iron deficiency put in fungal gene to break down phytate and a gene to store iron and to promote iron absorption Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied plants rice rich in: b-carotene iron added 6 genes from unrelated species Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied protein production if we know the DNA sequence we transform cells to make the protein human growth hormone, blood-clotting factors, insulin,… Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied protein production if we know the DNA sequence we transform cells to make the protein human growth hormone, blood-clotting factors, insulin,… Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Fig. 10.26. Relative numbers of patents issued for various clinical applications of the products of GE human genes. [Data from S. M. Thomas, et al., 1996. Nature 380: 387] © 2006 Jones and Bartlett Publishers 10.5 Genetic engineering applied gene therapy retroviruses remove “bad” viral genes put in “fixed” sequence virus will infect cell and insert its’ new RNA Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied gene therapy SCID severe combined immunodeficiency syndrome (non-functional immune system) Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5 Genetic engineering applied gene therapy SCID gene(s) identified - ADA remove bone marrow cells infect with retrovirus having fixed gene reinsert cells Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 4/10 developed leukemia 10.5 Genetic engineering applied vaccine production production of “natural” vaccines is often dangerous Fig. 10.10C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] The end Chapter 6 6.6 - 6.8 Practical applications of our knowledge of DNA structure Group worksheet Fig. 6.29. Structures of normal deoxyribose and the dideoxyribose sugar used in DNA sequencing © 2006 Jones and Bartlett Publishers Fig. 6.30. Dideoxy method of DNA sequencing. © 2006 Jones and Bartlett Publishers Fig. 6.30. Dideoxy method of DNA sequencing. © 2006 Jones and Bartlett Publishers G A T C (primer) 20 + Fig. 6.31. Florescence pattern trace obtained from a DNA sequencing gel © 2006 Jones and Bartlett Publishers