Gene cloning

• a collection of molecules or cells, all identical to an original molecule


  clone
• makes manipulating genes possible

  recombinant dna technology
• gene-sized pieces of dna in identical copies

  dna cloning
• basics of cloning


  source of dna

  vector

  restriction enzyme

  dna ligase

  host cell
• dna extracted from cells and purified

  genomic dna
• reverse transcription of mRNA

  cDNA
• dna using pcr


  amplified dna
• dna made artificially 


  synthetic dna
• medical applications, protein production

  genetic engineering
• carry ligated foreign gene into host cell, maintain foreign gene in host cell, replicate, pass into new cells during cell division, expressed the cloned foreign gene to make protein


  vector
• small circular dna molecule that replicate separately from the bacterial chromosome

  plasmids
• uses bacteria to make multiple copies of a gene

  gene cloning
• extrachromosomal dna found in bacteria/fungi, close circular dna molecules, can replicate autonomously, can be transferred to other cells by conjugation, can be integrated into the chromosome

  plasmid
• presence of one plasmid in a cell excludes other plasmids

  plasmid incompatibility
• carry genes that are not essential under normal conditions

  plasmid
• controlled by plasmid itself

  number of plasmid per cell
• high copy number plasmid

  pbr322
• important dna elements


  ori

  antibiotic resistant gene

  restriction sites
• destroy invading foreign dna, recognize specific short sequences of dna, cuts dna

  restriction enzyme
• two different enzymes have the same recognition sites,

  isoschizomers
• restriction enzymes restriction sites

  palindromic sequences
• restriction enzymes form?


  hairpin loops
• cuts asymmetrically


  sticky-end fragments
• cuts on line of symmetry

  blunt end fragments
• allows for proper reannealing and joining of dna molecules

  overhangs
• the cells must be made competent by treating with?

  cacl2
• the cells must be made transformed by undergoing?

  heat shock
• no vector


  will not grow on media + ampicillin
• vector


  will grow on media + ampicillin
• recombinant dna


  will grow on ampicillin
• to obtain transformants carrying cloned insert dna

  cloning
• use two different restriction enzymes to cut each end of the vector

  directional cloning
• cannot self ligate


  different sticky ends
• both the 3' OH and 5' PO4 are required for ligation, uses phosphatase


  dephosphorylation of vector
• multiple cloning site sequence is inserted into lac z


  lac z complementation
• to do blue white selection, the gene of interest is cloned into?

  multiple cloning site
• transformants are plated onto a medium containing:

  antibiotic for selection

  iptg to induce lac z

  x-gal to detect b-galactosidase
• transformants with vector only:

  alpha fragment

  fully active enzyme

  blue color colony
• transformants with recombinant dna


  no alpha fragment

  cannot form fully active enzyme

  white color colonies
• clone foreign gene and make foreign protein, requires extra dna elements

  expression vector