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© 2006 Jones and Bartlett Publishers
Chapter 10
Recombinant DNA Techniques10.1 cloning DNA- basics10.4 transgenic organisms - reverse genetics10.5 genetic engineering
10.1 Recombinant DNA Techniques
cut DNA with restriction enzymetake fragments
reassemble in new combinationsput back into organism (cell)
transgenic organism
(gene cloning)
10.1 Recombinant DNA Techniques
restriction enzymes
(gene cloning)
cut DNA at specific sequencesrestriction sites(palindromes)
10.1 Recombinant DNA Techniques
restriction enzymes
(gene cloning)
sticky ends5’ overhang3’ overhang
(complementary)blunt ends
© 2006 Jones and Bartlett Publishers
Fig. 10.2. Two types of cuts made by restriction enzymes
10.1 Recombinant DNA Techniques
restriction enzymes
(gene cloning)
5’-----GAATTC-----3’3’-----CTTAAG-----5’
3’ 5’5’-----GAATT C-----3’3’-----C TTAAG-----5’
5’ 3’
EcoRI
stick
y ends
10.1 Recombinant DNA Techniques
restriction enzymes
(gene cloning)
5’-----GAATTC-----3’3’-----CTTAAG-----5’
3 5’5’-----GAATTC-----3’3’-----CTTAAG-----5’
EcoRI
DNA ligase3’5’
5’3’
© 2006 Jones and Bartlett Publishers
Fig. 10.1. Circularization of DNA fragments produced by a restriction enzyme
10.1 Recombinant DNA Techniques
restriction enzymes
(gene cloning)
vectors
DNA sequence used to carry other DNA
10.1 Recombinant DNA Techniques
vectors
(gene cloning)
•can be put in a host easily•contains a replication origin•have a gene for screening
(eg. antibiotic resistance)
10.1 Recombinant DNA Techniques
vectors
(gene cloning)
•for E. coli - plasmids bacteriophage M13
© 2006 Jones and Bartlett Publishers
Fig. 10.5. Common cloning vectors for use with E. coli
10.1 Recombinant DNA Techniques
vectors
(gene cloning)
put into cells via
transformationelectroporation
© 2006 Jones and Bartlett Publishers
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
10.1 Recombinant DNA Techniques
DNA to insert ?
(gene cloning)
libraries
genomiccDNA
collections of vectors (lots)each containing cloned DNA
10.1 Recombinant DNA Techniques
genomic library (1)
(gene cloning)
phage
cut with restriction enzyme
x 10?
“sticky ends”
10.1 Recombinant DNA Techniques
genomic library (2)
(gene cloning)
cut with same restriction enzyme
“sticky ends”
10.1 Recombinant DNA Techniques
genomic library (3)
(gene cloning)
don’t forget DNA ligase
…lots of different vectors
10.1 Recombinant DNA Techniques
cDNA library
(gene cloning)
eukaryotic DNA has lots of intronsgenes are very large
if we are only interested in the partof the gene that codes for protein…
10.1 Recombinant DNA Techniques
cDNA library (1)
(gene cloning)
isolate the mRNA from the cell(s)
oligo-dT column
10.1 Recombinant DNA Techniques
cDNA library (2)
(gene cloning)
5’-----------------AAAAAA-3’ mRNA use reverse transcriptase
3’-----------------TTTTTTT-5’ DNA5’ -----------------AAAAAA-3’ DNA
then DNA polymerase… …a double stranded DNA from each mRNA
complementary DNA - cDNA
10.1 Recombinant DNA Techniques
cDNA library (3)
(gene cloning)
ligate DNAs into vectors
© 2006 Jones and Bartlett Publishers
Fig. 10.8. Reverse transcriptase produces a single-stranded DNA complementary in sequence to a template RNA
10.1 Recombinant DNA Techniques(gene cloning)
transformationor
electroporation
mix vectors (with insert) with cells
libraries
collections of vectors withdifferent DNA inserts
genomiccDNA
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. 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]
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 bacteriathat 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. 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]
More about plasmids
need to screen for bacteriathat 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. 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
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
grow on ampicillin with Xgal
© 2006 Jones and Bartlett Publishers
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
plasmid only
plasmid withinsert
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
Screening the library
106 to 10? of different clones
How do you “find” the one you want ?
© 2006 Jones and Bartlett Publishers
Fig. 10.11. Colony hybridization
© 2006 Jones and Bartlett Publishers
Chapter 10
Recombinant DNA Techniques10.1 cloning DNA- basics10.4 transgenic organisms - reverse genetics10.5 genetic engineering
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
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
Drosophila P elementsC. elegansmouse ESCdomestic animals
transforming the germ line
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.4 Reverse genetics
transposase
enzyme that can insert DNAflanked by inverted repeats
can place itself randomly into the chromosome
© 2006 Jones and Bartlett Publishers
Fig. 10.18. 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. 10.18. Transformation in Drosophila mediated by the transposable element P
your DNA + marker (eye color)
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.4 Reverse genetics
mouse
put DNA into fertilized eggusing engineered retrovirus
Embryonic stem cellsinsert modified cells into blastocyst
© 2006 Jones and Bartlett Publishers
Fig. 10.19. Transformation of the germ line in the mouse using embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.]
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.4 Reverse genetics
gene targeting
fig. 10.20
© 2006 Jones and Bartlett Publishers
Fig. 10.20. Gene targeting in embryonic stem cells. [After M.R. Capecchi. 1989. Trends Genet. 5: 70.]
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.4 Reverse genetics
Ti plasmid used on plantsAgrobactgerium
fig. 10.21
© 2006 Jones and Bartlett Publishers
Fig. 10.21. Transformation of a plant genome by T DNA from the Ti plasmid
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.4 Reverse genetics
Transformational rescue
fig. 10.22
by using inserts of different lengthsyou can find out how much of the DNA is necessary
© 2006 Jones and Bartlett Publishers
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]
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.5 Genetic 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.
http://www.nytimes.com/2007/07/30/washington/30animal.html?_r=1&oref=slogin
10.5 Genetic engineering applied
plants
increase nutritional value
-caroteneprecursor to vitamin Ain yellow vegetables
high rice diets of lack -carotene
Fig. 10.25 Rice engineered to produce -carotene
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.5 Genetic engineering applied
rice with:b-carotene
plants
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
rice rich in:b-caroteneiron
plants
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,…
© 2006 Jones and Bartlett Publishers
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]
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.5 Genetic engineering applied
gene therapy
retroviruses
remove “bad” viral genesput in “fixed” sequencevirus 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 immuno- deficiency 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 - ADAremove bone marrow cellsinfect with retrovirus having
fixed genereinsert cells
4/10 developed leukemia
Fig. 10.10C. Transformed bacterial colonies.[Courtesy of Elena R. Lozovsky]
10.5 Genetic engineering applied
vaccine production
production of “natural” vaccines is often dangerous
The end
Chapter 6
6.6 - 6.8 Practical applications of ourknowledge of DNA
structureGroup worksheet
© 2006 Jones and Bartlett Publishers
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.
G A T C
(primer) 20 +
© 2006 Jones and Bartlett Publishers© 2006 Jones and Bartlett Publishers
Fig. 6.31. Florescence pattern trace obtained from a DNA sequencing gel
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