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MBIO 3410 lecture Oct 25 2007

MBIO 3410 lecture Oct 25 2007 - Last Lecture Overview...

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1 Last Lecture Overview Bacteriophage vectors Bacteriophage λ λ genome Lysogenic pathway Lytic pathway Early transcription Delayed early transcription Replication Late transcription in vivo packaging of λ DNA Formation of plaques λ as a vector Types of λ vectors Insertional vector λ replacement vectors Strategies to identify λ phage recombinants Inactivation of the cI gene Blue–white screening in vitro λ packaging λ lysogens M13 phage vectors Cloning in M13
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2 Phagemids
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3 Phagemids Phagemids are plasmids that contain the f1 phage origin of replication for the production of single-stranded DNA Phagemids are generally small plasmids can accept larger DNA inserts than M13-based vectors f1 origin of replication can be cloned into pBR322 however it is not sufficient to direct single-stranded DNA production A bacterium carrying a phagemid infected with a functional wild-type M13 or f1 helper phage produces single-stranded phagemid DNA The phagemid single-stranded DNA would be packaged into viral particles and secreted into the surrounding medium in the same way that M13 phage particles are produced Cloning the f1 origin in the reverse orientation leads to the production of the opposite strand of DNA In the absence of a helper phage double-stranded DNA can be isolated as a normal plasmid
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4 pBluescript phagemid vector pBluescript is a plasmid vector that has been developed to incorporate M13 functionally: Contains both plasmid and M13 origin of replication Lack genes required for the full phage lifestyle So they are propagated as a true plasmid and have the advantage of rapid replication and growth and can be induced to produce single stranded phage particles by co-infection with a fully functional helper phage Provides the gene products required for single strand production and packaging
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5 Cloning large DNA fragments
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6 Cloning large DNA fragments The analysis of genome organization and identification of genes in organisms with large genome sizes is difficult using plasmid and bacteriophage λ vectors which contain a maximum size range Due to the size constraints an enormous number of clones would be required to represent the whole genome in a DNA library Some eukaryotic genes contain large intron sequences making more difficult to fit the entire gene into a single cloned fragment Alternative methods to conventional vectors can be used to generate libraries from large genomes: Cosmids Artificial chromosomes YACs PACs BACs HACs
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7 Cosmids
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