Module 10: Genetic Engineering

• molecular cloning = cutting and joining propagating recombinant DNA
• Uses for isolating genes:

  1. gene libraries
  2. cDNA
  3. Genomic
• cloning DNA fragments:

  1. reduces the complexity of DNA
  2. allows large scale production and analysis of purified single sequences
• Molecular cloning overview:

  1. isolate DNA
  2. Cut DNA
  3. join to a vector (recombinant)
  4. introduce recombine vector into bacteria (transformation)
  5. amplify recombination DNA by bacterial growth
• Cutting and Splicing DNA

  *restriction endonuclease

  *cuts plaindromic sequence

  *sticky ends can be rejoined

  *there are many restriction endonuclease, each with different sequence specifities
• 1. Cut DNA fragment out of plasmas via DNA ligase and phosphatase
  2. introduce into E.coli and select for antibiotic resistance
  3. circularised DNA fragment will not replicated
  4. unmofidised plasmid- phosphate prevents he recircularisation of the plasmid
  5. recombinant plasmid is desirable
• DNA libraries- isolation and separation of individual sequences within a cell (nuclear DNA = genomic library)
• cDNA libraries hold mRNA

  *only expressed genes

  *(tissue) specific cell types

  *must be converted to DNA to be cloned (complementary or cDNA)
• cDNA

  1. isolate mRNA
  2. convert to cDNA- via reverse transcriptase
  3. insert into a vector and transform
  4. colonies (clones)- 1 sequence per colony
• Genomic

  1. Isolate DNA
  2. cut DNA
  3. insert into vector
• Disadvantages of cDNA:

  *comprises expressed genes (transcriptome)

  *'libraries' from different tissues contain different sequences

  *no untranscribed sequences
• Disadvantages of Genomic:

  *comprises sequences representing the genomes- same in all tissues

  *includes intron sand regulatory sequences as well as exons

  *'Raw material' for gene mapping and genome projects
• Therapeutic proteins

  1. Fuse coding region of gene to a strong promoter
  2. insert recombinant gene into host
  3. host multiplies and overproduces protein
  4. purify protein
• HOST SYSTEMS- Bacteria

  Advantages:

  1. cheap
  2. fast growing
  3. easy to maintain
  4. stable

  Disadvantages:

  1. proteins may be insoluble/ denatured
  2. no post- translational modification
• HOSY SYSTEMS- animal cells

  Advantages:

  1. post-translational modification
  2. soluble/ properly folded

  Disadvantages:

  1. expensive
  2. unstable
• Case study- Insulin

  *only obtained via animal sources

  *can produce human insulin in bacteria

  *insulin is processed

  *easier to synthesise 2 chains apart then join