1. Restriction enzymes (endonucleases)
Cleave a specific DNA sequence
Protect bacteria from phage infection by digesting phage DNA after infection.
Cellular DNA is protected by methylases - block restriction enzyme activity

2. Each organism has a specific set of restriction enzymes:
EcoRI from Escherichia coli
BamHI from Bacillus amyloliqueraciens
PvuI and PvuII are different enzymes from same strain.
Originally purified by individual labs
Now supplied by companies - NEB, Promega, BRL
Ch. 3-1

3. Restriction enzymes are used for cloning and analyzing DNA fragments
Used to excise specific fragments of DNA from a plasmid RI/HindIII from A, clone into B
Can then paste them back together (ligate) - get desired clone but will also get other combinations
Then use restriction enzymes to screen for the desired plasmid
Can screen for the presence of multiple PstI restriction sites. Only in desired clone

4. Sequence Recognition and cleavage:
a) 5' overhang EcoRI GAATTC G AATTC
CTTAAG CTTAA G
b) 3' overhang KpnI GGTACC GGTAC C
CCATGG C CATGG
c) Blunt end SmaI CCCGGG CCC GGG
GGGCCC GGG CCC
Different types of overhangs
Need compatible ends with same overhangs
except for blunts, can ligate any two blunts
Blunt ligations are harder, no overlap for base pairing, not very sticky
Ch. 3-2

5. Sticky ends
The overhangs on cleaved DNA can serve as “sticky ends” or unpaired bases that can be used to link pieces of DNA.
Use the same enzyme, or one that leaves the same overhang to cut two DNA sources.
Complementary bases will pair.
Ligase will seal.

6. Vectors
In order to study a DNA fragment (e.g., a gene), it must be amplified and eventually purified.
Do this by cloning the DNA into a vector, generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli.
Ch. 1-1

7. Cloning Scheme
Digest
Ligate
Amplify and Prep
1-1

8. Vector Types There are three commonly used types of vectors:
1) plasmid vectors (e.g., pUC plasmids)
These are the most common in biotechnology
2) bacteriophage vectors (e.g., phage )
3) phagemid (hybrid) vectors
Each has a different use, and there are many derivatives of these basic building blocks.
Ch. 1-1

9. Plasmids Circular DNA molecules found in bacteria
Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication.
Some plasmids are present in E. coli at copies/cell

10. Plasmid Engineering Plasmids also contain selectable markers:
These are genes encoding proteins which provide a way to rapidly and easily find bacteria containing the plasmid.
Commonly- provide resistance to an antibiotic like ampicillin.
Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker.
Ch. 1-2

11. Safety Features
Modern cloning plasmids have been engineered to be incapable of transfer between bacterial cells
Provide a level of biological containment.
Naturally occurring plasmids with their drug resistance genes have produced antibiotic-resistant bacteria.
Ch. 1-2

12. Transforming plasmids
Into bacteria
Ch. 1-2

13. Screening for Inserts
Transform bacteria with plasmids containing gene for ampicillin resistance; small number will transform.
Spread bacteria on plates containing nutrient agar and ampicillin.
Only transformed cells will survive and form colonies.
1-3