Sex

1
Sex!
2
Bacteria Sex
3
Transformation
Transformation DNA picked up directly from the
medium and recombined into the genome
4
Transduction
5
Plasmids
6
Conjugation
7
Resistance Plasmids
8
Genetic Engineering
9
Restriction Endonucleases

• A Restriction Endonucleases will cut both strands
  of a DNA duplex at a specific place
• These places need not be directly opposite

5GAATTC3
3CTTAAG5
5G -OH P-AATTC3
3CTTAA -P HO-G5

• Note that this enzyme used is EcoRI, the first
  restriction endonuclease characterized

10
Sticky Ends
11
Ligation
12
More RE Enzymes
13
Most ER RecognitionSequences are Palindromes
GAATT-C C-TTAAG
GGATC-C C-CTAGG
Nodeba Bob Abedon
AGATC-C T-CTAGG
GCGGCC-GC CG-CCGGCG
14
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
15
Ligation into Plasmid
Upon Ligation we now have Recombinant DNA!
16
Transformation
Note that plasmid is vector that carries DNA into
recipient cells
Other vectors include viruses (transduction) as
well as otherwise inert projectiles
17
Virus (Phage) Vector
18
Clone Identification
19
Now What?
20
Polymerase Chain Reaction
21
Gel Electrophoresis (1/2)
22
Gel Electrophoresis (2/2)
23
Southern Blotting
24
Restriction Fragment Length Polymorphism
25
DNA Sequencing
26
DNA Microarrays
27
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
28
Link to Next Presentation
29
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//w
ww.ipam.ucla.edu/publications/fg2000/fgt_tspeed8.p
pt http//www.bioscience.drexel.edu/Homepage/Fall2
002/Bio449/powerpoints/BIO44920lab20lecture203.
ppt