BIO2015: Lecture 3

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  • 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

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