ISOLATION

• HOW TO MAKE PROTEINS USING GENETIC TECHNOLOGY?

  1. Isolation of DNA fragments of gene of interest
  2. Insertion of DNA fragment into vector
  3. Transformation (transferring to host cell)
  4. Identification of successful host cells using gene marker
  5. Growth/ cloning of population of host cells
• METHODS OF ISOLATION?

  1. Reverse transcriptase
  2. Restriction endonucleases
  3. Gene machine
• REVERSE TRANSCRIPITASE PROCESS?

  1. Take mRNA coding for gene
  2. Add reverse transcriptase
  3. Produces cDNA
  4. Add enzyme to separate cDNA and mRNA
  5. Add DNA polymerase
  6. Produces double stranded DNA of gene
• REVERSE TRANSCRIPTASE EVALUATION?

  + mRNA easier to obtain than DNA

  + cheap

  -must identify correct mRNA
• RESTRICTION ENDONUCLEASES PROCESS?

  • cuts DNA double sequence at specific base sequence (recognition site)
  • cuts between two opposite base pairs
  • produces straight edges/ blunt ends
  • or cuts in staggered fashion
  • produces uneven cut (each strand has exposed, unpaired bases)/ sticky ends
• RESTRICTION ENDONUCLEASES EVALUATION?

  + cheap

  + useful sticky ends produced

  -DNA must be removed from nucleus

  -restriction endonucleases must be found which cut before and after gene
• GENE MACHINE PROCESS?

  1. Desired sequence of nucleotide bases for gene fed into computer
  2. Computer produces oligonucleotides (small, overlapping single strands of DNA)
  3. Joined together to produce a gene (with no introns or non-coding DNA)
  4. Complementary strand made
  5. Gene copied
  6. Can be inserted into bacterial plasmid using sticky ends
• GENE MACHINE EVALUATION?

  + quick

  + accurate

  + no introns so no splicing and can be directly inserted into prokaryotes

  -expensive technology