MBIO 3410 lecture Oct 30 2007

MBIO 3410 lecture Oct 30 2007 - Last Lecture Overview...

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1 Last Lecture Overview Phagemids pBluescript phagemid vector Cloning large DNA fragments Cosmids Artificial Chromosomes YACs A yeast centromere Yeast autonomously replicating sequence Yeast telomeres Genes for YAC selection in yeast Bacterial replication origin and a bacterial selectable marker YAC Construction Selection Difficulties associated with YACs
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2 PACs
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3 PACs (P1 artificial chromosome) To overcome problems associated with using cosmid or YAC systems, a method for cloning and packaging DNA fragments using a bacteriophage P1 system offers the ability to clone large genomic DNA fragments of between 70-95 kb in size P1 bacteriophage (larger genome than λ phage) vectors have been designed with the essential replication components of P1 incorporated into a plasmid Upon infecting E. coli , P1 may either: Express lytic functions = producing 100–200 new bacteriophage particles and lysing the infected bacterium Bacteriophage may repress its lytic functions maintaining the bacteriophage genome as a large, stable, low-copy plasmid
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4 P1 phage has two replication origins: One controls lytic DNA replication the other maintains the plasmid during non-lytic growth During the lytic cycle, new phage DNA is produced and cleaved at a pac site prior to insertion into phage particles
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5 Cloning with P1 vectors The PAC vector is digested with the restriction enzymes Sca I and Bam HI to generate two vector arms: a short and a long arm Genomic DNA is partially digested with Mbo I yielding BamH I-compatible sticky ends Fragments between 70-95 kb in length are isolated and ligated between the vector arms to generate a series of linear molecules
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6 If ligation occurs between two short arms the resulting molecule will contain neither the P1 replication origins nor the KAN R gene will be non-viable If both arms are long there will be no pac site no packaging into the phage heads will occur The only viable recombinant will consist of the insert sequence flanked by both a short and long arm
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7 Phage P1 uses a ‘head-full’ packaging strategy and can accommodate a total DNA length of approximately 110-115 kb This means that any inserts longer than 95–100 kb will result in the truncation of the packaged DNA before both lox P sites are inserted into the phage and the molecule will be unable to circularize upon transfection into the host Once injected into the cre + E. coli host cell, the Cre protein circularizes the DNA at the lox P sites and DNA then replicates using the plasmid origin of replication
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8 The original vector BamHI restriction enzyme site, into which the foreign DNA was inserted, is located within the bacterial sacB gene (encoding levansucrase) The expression of this gene is toxic to E. coli cells growing on sucrose Thus sucrose growth provides a mechanism of positive selection for those PACs containing inserts.
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