Lecture 3 Genetically Engineering Organisms

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Dec 14, 2012 (4 years and 8 months ago)

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Lecture 3

Genetically Engineering
Organisms

Terms


Genetic Engineering




set of tools not a discipline




manipulation of DNA outside the cell to create artificial
genes or novel combinations of genes with predesigned
control elements.


Recombinant DNA


(plasmid movie)


a general term for the laboratory manipulation of DNA
in which DNA molecules or fragments from various
sources are severed and combined enzymatically and
reinserted into living organisms.

Tools


Restriction Enzymes

cut DNA movie


Restriction Enzymes
-

recognize specific short
sequences of DNA and cleave the DNA at that site
or near the recognition site (4 and 8 base pairs)



EcoRI
-

E. coli
-

5'

G>AATTC 3'






CTTAA<G


EcoRV
-

E. coli
-


GAT<ATC






CTA<TAG


Hae II
-

Haemophilus aegyptius


Tth111 I
-

Thermus thermophilius III

GACN<NNGTC





CTGN<NNCAG


Tools


DNA ligase
-

enzyme which permanently joins
DNA ends together


DNA polymerase
-

fills in 5' to 3' direction, 3' to
5' exonuclease (cuts bases off)


Linkers
-

short fragments of DNA 10 bp with a
restriction site



Vector
-

a plasmid or phage that is used to "carry"
inserted foreign DNA for the purpose of
producing more material or a protein product


Usually contains antibiotic resistance gene


Operator and promoter like tac for bacteria


Multiple cloning site
-

contains between 6 and 12
unique restriction fragments for cloning



Tools


CAP
-

calf alkaline phosphatase
-

catalyzes the
hydrolysis of 5' phosphate groups on DNA,
prevent recirculation of vectors


Movie


20
-
03 cloning


Cut chromosome with restriction enzyme,
recombine fragments with ligase enzyme in a
vector and insert into host (transformation) scan
for correct gene




Scanning for Correct Gene


Plate on media with antibiotic


If using tac promoter


use blue/white colony
selection and special media



Hybridization
-

have a similar piece of DNA
(partial)
-

probe
-

radiolabel it and check to see
which clones it reacts with.


lyse and transfer on nitrocellulose


requires pre knowledge of part of gene have one from
another organism want to find in new or know amino
acid sequence chemically synthesis gene and make
probe


Check for protein expression.


Movie


Chap 14 1and 2



Higher Organisms


more difficult due to lack of transformation
techniques


lack of understanding of the host cell
genetics


Continuously gaining knowledge


Plant Examples


Infect with a bacterium
Agrobacterium
tumefaciens
.


Bacterium carries a plasmid with DNA that can be
incorporated into the plant chromosome(T DNA)



not many plants can be infected with this bacterium


Biolistic process


coat small projectiles (1 micron with tungsten) with
DNA


shoot these into cells at a high velocity.


Higher Organism Examples


Electroporation



exposing cells to a brief high voltage electri discharge
which renders the cells permeable to DNA.


Used with fungus, bacteria, animals, and plant cells.



Microinjection


Pipette and microscope


inject directly into
microscope


Mammalian cells


modify virus to infect cells


baculovirus from insects
-

used to place a desired gene
under the control of a strong promoter.



Higher Organisms


Gene
Transfer


Why?


Combat disease


Genetic defects


new borns


Single Dose Cure


Cell transplantation


HIV 1, Rheumatoid Arthritis


Table 11.1

Permanent Gene Manipulation


Not what gene transfer therapy is about


Somatic cell gene therapy


only affects the
recipient


Germ cell gene therapy


Affects fertilized cell


Inherited in next generation


Not legal in US

Retrovirus Gene Transfer


Retrovirus cycle


(movie chap 18 3)

Retrovirus


Replication incompetent


so virus does not
replicate


Remove viral genes from virus’ genonome


Substitute therapeutic genes


Figure 11.3 in text


RNA transfer

HIV Virus

Producing Replication
-
incompetent retrovirus

Production
-

continued

Adenovirus


Transfer virus DNA


Can produce upper respiratory infections


Can’t integrate into host cell’s genonome


Safe when administered to humans


Package like retrovirus Figure 11.5


Homologous recombination in special
vector


High expression of genes

Comparison


Table 11.3


Compares Adenovirus and Retrovirus

Gene Amplification


Why?


Sequencing


Detecting disease


PCR


polymerase chain reaction
-

movie


in vitro method of amplifying DNA sequences beginning
with DNA from any source, bacteria, plant etc.


need to know the sequence (20 bp) around the gene of
interest
-

both sides
-

synthesis this
-

primer


apply heat to separate strands of DNA


add primers and bases (ATCG) and an enzyme Taq
polymerase
-

complementary DNA is synthesized


quick reaction (n cycles 2
n

times the amount of DNA) Can
do 30 cycles per day 2
30

copies of the gene
-

billion fold
increase


Chap 14 6


Microarrays


Movie Chap 14 4


Chap 15 1 and 2