© 2006 Jones and Bartlett Publishers Chapter 10 Recombinant DNA Techniques 10.1cloning DNA- basics 10.4transgenic organisms - reverse genetics 10.5genetic engineering

10.1Recombinant DNA Techniques cut DNA with restriction enzyme take fragments reassemble in new combinations put back into organism (cell) transgenic organism (gene cloning)

10.1Recombinant DNA Techniques restriction enzymes (gene cloning) cut DNA at specific sequences restriction sites (palindromes)

10.1Recombinant DNA Techniques restriction enzymes (gene cloning) sticky ends 5’ overhang 3’ overhang (complementary) blunt ends

© 2006 Jones and Bartlett Publishers Fig Two types of cuts made by restriction enzymes

10.1Recombinant DNA Techniques restriction enzymes (gene cloning) 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ 3’ 5’ 5’-----GAATT C-----3’ 3’-----CTTAAG-----5’ 5’3’ EcoRI sticky ends

10.1Recombinant DNA Techniques restriction enzymes (gene cloning) 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ 3 5’ 5’-----GAATTC-----3’ 3’-----CTTAAG-----5’ EcoRI DNA ligase 3’5’ 5’3’

© 2006 Jones and Bartlett Publishers Fig Circularization of DNA fragments produced by a restriction enzyme

10.1Recombinant DNA Techniques restriction enzymes (gene cloning) vectors DNA sequence used to carry other DNA

10.1Recombinant DNA Techniques vectors (gene cloning) can be put in a host easily contains a replication origin have a gene for screening (eg. antibiotic resistance)

10.1Recombinant DNA Techniques vectors (gene cloning) for E. coli - plasmids bacteriophage M13

© 2006 Jones and Bartlett Publishers Fig Common cloning vectors for use with E. coli

10.1Recombinant DNA Techniques vectors (gene cloning) put into cells via transformation electroporation

© 2006 Jones and Bartlett Publishers Fig Construction of recombinant DNA plasmids containing fragments derived from a donor organism

© 2006 Jones and Bartlett Publishers Fig Example of cloning

10.1Recombinant DNA Techniques DNA to insert ? (gene cloning) libraries genomic cDNA collections of vectors (lots) each containing cloned DNA

10.1Recombinant DNA Techniques genomic library (1) (gene cloning) phage cut with restriction enzyme x 10 ? “sticky ends”

10.1Recombinant DNA Techniques genomic library (2) (gene cloning) cut with same restriction enzyme “sticky ends”

10.1Recombinant DNA Techniques genomic library (3) (gene cloning) don’t forget DNA ligase …lots of different vectors

10.1Recombinant DNA Techniques cDNA library (gene cloning) 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…

10.1Recombinant DNA Techniques cDNA library (1) (gene cloning) isolate the mRNA from the cell(s) oligo-dT column

10.1Recombinant DNA Techniques cDNA library (2) (gene cloning) 5’ AAAAAA-3’ mRNA use reverse transcriptase 3’ TTTTTTT-5’ DNA 5’ AAAAAA-3’ DNA then DNA polymerase… …a double stranded DNA from each mRNA complementary DNA - cDNA

10.1Recombinant DNA Techniques cDNA library (3) (gene cloning) ligate DNAs into vectors

© 2006 Jones and Bartlett Publishers Fig Reverse transcriptase produces a single-stranded DNA complementary in sequence to a template RNA

10.1Recombinant 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)

© 2006 Jones and Bartlett Publishers Fig (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]

AATTC- our - DNA -A G- our - DNA -TTCGA More about plasmids (directional cloning) …G …CTTAA AATTCGATATCA GCTATAGTTCGA AGCTT… A… EcoRI HindIII AATTC- our - DNA -A G- our - DNA -TTCGA

More about plasmids need to screen for bacteria that with the plasmid need to have lots of different single site RE sites you only want to grow the bacteria took up the plasmid

© 2006 Jones and Bartlett Publishers Fig (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]

More about plasmids need to screen for bacteria that with the plasmid need to screen for plasmids with an insert need to have lots of different single site RE sites some will have closed up without insert

© 2006 Jones and Bartlett Publishers Fig A,B. Detection of recombinant plasmids through insertional inactivation of a fragment of the lacZ gene from E. coli

© 2006 Jones and Bartlett Publishers Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] grow on ampicillin with Xgal

© 2006 Jones and Bartlett Publishers Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] plasmid only plasmid with insert

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] Screening the library 10 6 to 10 ? of different clones How do you “find” the one you want ?

© 2006 Jones and Bartlett Publishers Fig Colony hybridization

© 2006 Jones and Bartlett Publishers Chapter 10 Recombinant DNA Techniques 10.1cloning DNA- basics 10.4transgenic organisms - reverse genetics 10.5genetic engineering

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics In the past… find mutant phenotype find mutant gene study wild-type gene

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics but now we can… mutate a gene find study the phenotype

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics DrosophilaP elements C. elegans mouseESC domestic animals transforming the germ line

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics transposase enzyme that can insert DNA flanked by inverted repeats can place itself randomly into the chromosome

© 2006 Jones and Bartlett Publishers Fig Transformation in Drosophila mediated by the transposable element P remove some of the inverted repeats -cannot be inserted and insert DNA into coding region

© 2006 Jones and Bartlett Publishers Fig Transformation in Drosophila mediated by the transposable element P your DNA + marker (eye color)

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics mouse put DNA into fertilized egg using engineered retrovirus Embryonic stem cells insert modified cells into blastocyst

© 2006 Jones and Bartlett Publishers Fig Transformation of the germ line in the mouse using embryonic stem cells. [After M.R. Capecchi Trends Genet. 5: 70.]

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics gene targeting fig

© 2006 Jones and Bartlett Publishers Fig Gene targeting in embryonic stem cells. [After M.R. Capecchi Trends Genet. 5: 70.]

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics Ti plasmid used on plants Agrobactgerium fig

© 2006 Jones and Bartlett Publishers Fig Transformation of a plant genome by T DNA from the Ti plasmid

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.4Reverse genetics Transformational rescue fig by using inserts of different lengths you can find out how much of the DNA is necessary

© 2006 Jones and Bartlett Publishers Fig Genetic organization of the Drosophila gene white

© 2006 Jones and Bartlett Publishers Fig Eyes of a wildtype red-eyed male D. melanogaster and a mutant white-eyed male. [Courtesy of E. Lozovsky]

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied Animal growth rate metallothionen promoter (very active) growth hormone

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.

10.5Genetic engineering applied plants increase nutritional value  -carotene precursor to vitamin A in yellow vegetables high rice diets of lack  -carotene

Fig Rice engineered to produce  -carotene

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied rice with: b-carotene plants

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic 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 C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied rice rich in: b-carotene iron plants added 6 genes from unrelated species

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic 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 C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied protein production if we know the DNA sequence we transform cells to make the protein human growth hormone, blood-clotting factors, insulin,…

© 2006 Jones and Bartlett Publishers Fig Relative numbers of patents issued for various clinical applications of the products of GE human genes. [Data from S. M. Thomas, et al., Nature 380: 387]

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied gene therapy retroviruses remove “bad” viral genes put in “fixed” sequence virus will infect cell and insert its’ new RNA

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied gene therapy SCID severe combined immuno- deficiency syndrome (non-functional immune system)

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied gene therapy SCID gene(s) identified- ADA remove bone marrow cells infect with retrovirus having fixed gene reinsert cells 4/10 developed leukemia

Fig C. Transformed bacterial colonies. [Courtesy of Elena R. Lozovsky] 10.5Genetic engineering applied vaccine production production of “natural” vaccines is often dangerous The end

Chapter Practical applications of our knowledge of DNA structure Group worksheet

© 2006 Jones and Bartlett Publishers Fig Structures of normal deoxyribose and the dideoxyribose sugar used in DNA sequencing

© 2006 Jones and Bartlett Publishers Fig Dideoxy method of DNA sequencing.

© 2006 Jones and Bartlett Publishers Fig Dideoxy method of DNA sequencing.

G A T C (primer) 20 +

© 2006 Jones and Bartlett Publishers Fig Florescence pattern trace obtained from a DNA sequencing gel