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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