BIO2015: Lecture 3
- Created by: LMoney
- Created on: 08-04-14 13:45
Constructing DNA libraries with λ phage and other cloning vectors
· Cloning all of genomic DNA of higher organisms into plasmid vectors- not practical due to relatively low transformation efficiency of E.coli and small number of transformed that can be grown on typical culture plate
· Cloning vectors derived from bacteriophage- no such limitations- bacteriophage are viruses that infects and replicates within bacteria
· Collection of clones that includes all DNA sequences of given species- genomic library
· Genomic library can be screened for clones containing sequence of interest
bacteriophage λ undergoes either lytic replication or lysogeny following infection of E.coli
lytic replication of bacteriophage λ results in cell death of E.coli and formation of clear plaques on cell lawn
· heads and tails of λ are systems of self assembling proteins
· addition of DNA containing COS sites at correct spacing (43-53kb) allows spontaneous formation of complete, functional λ phage- basis of in vitro packaging system
· preassembled λ head and concatomer of λ DNA combined
· Nu1 and A proteins promote filling of λ head with DNA between COS sites
· Λ genome (1 copy) is contained in preassembled λ head
· Preassembled λ tail is then attached because λ attaches only to filled head
Replication and packaging of bacteriophage lambda DNA
· COS site- rolling circle mode of DNA replication
· Endonuclease A cleaves at COS sites
· Lambda proteins assembled
· DNA packaged in phage head- upper limit 53kb
· Combined give infective particles
λ phage vectors
· Generally have central stuffer fragment- replaced by DNA to be cloned
· Central stuffer fragment contains genes used in lysogenic phase of the life cycle- i.e. causes phage to integrate into host genome and maintain integrated state
· Λ cloning vectors thus generally only undergo lytic part of life cycle
· When replacing stuffer fragments- DNA fragments to be cloned must allow functional phage to be produced- there is thus restricted size range of fragments that can be accepted
· Vectors designed for cloning genomic DNA generally accept fragments in range 12-20kbp
· Vectors designed for cloning cDNA accept fragments in range 0-10kbp
· Efficiency of host infection (transfection) is not dependent on insert size, provided that phage in correct size range results
Comparison of plasmid and phage cloning vectors
· Plasmid vectors:
· Circular DNA
· DNA to be cloned- inserted into vector
· Transformation…
Comments
No comments have yet been made