1. Topics to be covers Basic features present on plasmids
Eukaryotic and prokaryotic plasmids
lacZ complementation (blue white screening)
Replicative and integrative plasmids
Yeast artificial chromosome

2. DNA Plasmids
plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA
double-stranded and, in many cases, circular
Typically present in bacteria and lower eukaryotes
In molecular biology replicative and integrative vectors are used

3. Plasmids as Cloning Vectors
Cloning vector plasmid pUC19
Essential features include:
an ampicillin resistance marker
polylinker with multiple restriction enzyme cut sites.
Insertion of cloned DNA within the polylinker inactivates the truncated lacZ’ gene that encodes part of beta- galactosidase so that transformants with insert will be by white and cells without insert - blue

4. Principle of the lacZα complementation

5. Prokaryotic Expression Systems
Most often E. coli for screening of libraries
Prokaryotic promoter— ribosome binding site— MCS—transcription termination site
Prokaryotic selectable marker
Origin of replication
ori

6. Eukaryotic expression vectors
E. coli origin of replication
A selectable eukaryotic marker gene
A eukaryotic promoter sequence
The appropriate eukaryotic transcriptional and translational stop signal
A sequence that signals polyadenylation of the transcript messenger RNA (mRNA)
Origin of replication
Marker gene and ori for propagation of vector in E. coli
Eukaryotic marker system
Eukaryotic origin of replication
Coding sequence

7. Yeast artificial chromosome (YAC)
The YAC is designed to clone large fragments of DNA ( kb)
The YAC is maintained as a separate chromosome in the host yeast cell and is highly stable
Use of YAC:
the physical mapping of human genomic DNA
the analysis of large transcription units
the formation of genomic libraries containing DNA from individual human chromosomes

8. Construction of yeast artifical chromosomes (YAC)
Telomere sequence required to insure stability of YAC ends
DNA fragment can range from 100 to 1000kb

9. Integrative plasmids