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Sex!

Transformation
Transformation: DNA picked up
directly from the medium and
recombined into the genome

heteroduplex

Competent cell:
capable of picking
up DNA
Transduction

Viral products produced, host


genome fragmented

~1 phage/10,000 will
pick up chromosomal
DNA...
Plasmids
Conjugation
Resistance Plasmids
Genetic Engineering
Restriction Endonucleases
• A Restriction Endonucleases will cut both
strands of a DNA duplex at a specific place
• These “places” need not be directly opposite:

5’…GAATTC…3’
3’…CTTAAG…5’

5’…G -OH5’…GAATTC…3’
P-AATTC…3’

3’…CTTAA3’…CTTAAG…5’
-P HO-G…5’

• Note that this enzyme used is EcoRI, the first


restriction endonuclease characterized
Sticky Ends
Ligation
More RE Enzymes
Enzyme Sequence Product
EcoRI G^AATTC 5’ sticky ends
BamHI G^GATCC 5’ sticky ends
Bg1II A^GATCT 5’ sticky ends
PvuI CGATC^G 3’ sticky ends
PvuII CAG^CTG Blunt end
MboI G^ATC 5’ sticky ends
HindIII A^AGCTT 5’ sticky ends
HinfI G^ANTC 5’ sticky ends
Sau3A G^ATC 5’ sticky ends
AluI AG^CT Blunt end
TaqI T^CGA 5’ sticky ends
HaeIII G^GCC 5’ sticky ends
NofI GC^GGCCGC 5’ sticky ends
Most ER Recognition
Sequences are Palindromes

G^AATT-C G^GATC-C
C-TTAA^G C-CTAG^G
Nodeba Bob Abedon
A^GATC-C GC^GGCC-GC
T-CTAG^G CG-CCGG^CG
Complementary (c)DNA Note that the reverse
transcriptase is primed
by the poly-A found at
the end of most
eukaryotic mRNAs
Note that there are
issues (& variation) as
to how one makes the
second DNA strand
Here that strand is
generated using a
second enzyme that is
primed via generation
of a hairpin of DNA by
reverse transcriptase
Ligation into Plasmid

Upon Ligation we now have


Recombinant DNA!
Note that
plasmid is vector
Transformation
that carries DNA
into recipient
cells

Other vectors
include viruses
(transduction) as
well as
otherwise inert
projectiles
Virus (Phage) Vector
Polymerase Chain Reaction
Gel Electrophoresis (1/2)
Gel Electrophoresis (2/2)
Southern Blotting

Visualized DNA
Restriction Fragment Length Polymorphism
DNA Sequencing
DNA Microarrays
Monoclonal Antibodies (Mabs)

Note that though clonal, Monoclonal


Antibodies are (or at least used to be not)
produced by processes that have
nothing to do with Gene Cloning
Acknowledgements

http://www.healthsci.utas.edu.au/hls/units/cxa262/lectures/cloning.ppt
www.healthsci.utas.edu.au/hls/units/ cxa262/lectures/cloning.ppt
www.ou.edu/cas/zoology/Courses/3333/powerpoint/lec8.ppt
http://www.ipam.ucla.edu/publications/fg2000/fgt_tspeed8.ppt
http://www.bioscience.drexel.edu/Homepage/Fall2002/Bio449/powerpoints/BIO449%20lab%20lecture%203.ppt

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