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