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Manipulating DNA

DNA manipulation - uses enzymes (imitates what cells can do) 

  • restriction endonuclease - able to cleave DNA at specific places
    • restriction sites - where nucleases cleave DNA
    • methylation - stops nucleases from cleaving DNA
    • Type I - makes simple cuts on both DNA strands
    • Type II - makes staggered cuts where sequences same on both sides (dyad symmetry)
  • ligase - makes phosphodiester bonds to connect hydroxyl/phosphate groups
    • also joins Okazaki fragments on lagging strands
    • creates recombinant molecules from fragments created by nucleases

vector systems - used to carry recombinant DNA molecule into a cell 

  • not required by the cell, but can be selected w/ addition of marker
  • plasmids - small extrachromosomal DNA
    • must have origin of replication, selectable marker (usually for antibiotic resistance)
    • markers - used to see which cell took in the new DNA
    • multiple cloning site (MCS) - region in plasmid where DNA is inserted
    • inactivation of gene signals plasmid’s acceptance of new DNA
  • phages - viruses that infect bacterial cells
    • larger than plasmids, can insert more DNA
    • needs other live cells to replicate
    • linear DNA (can’t infect unless new DNA gets inserted)
  • chimera - totally new genome, nonexistent in nature
  • yeast artificial chromosome (YAC) - able to introduce larger DNA pieces than plasmids

DNA library - collection of all DNA fragments representing all an organism’s DNA 

  • genomic library - simplest type of DNA library
    • randomly fragmented genome
    • hydrodynamic shear forces - passes DNA through syringe
  • cDNA libraries - set of all expressed genes
    • reverse transcriptase - retrovirus that makes DNA from mRNA
Subject: 
Biology
Subject X2: 
Biology

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