INSERTION

• HOW IS A GENE PREPARED FOR INSERTION?

  • promoter added upstream of gene
  • provides binding site of RNA polymerase which begins transcription
  • terminator added downstream of gene
  • releases RNA polymerase which ends transcription
• PROCESS OF INSERTION?

  1. Use restriction endonuclease to produce sticky ends to cut genes out DNA
  2. Use same restriction endonucleases to cut plasmid
  3. Open plasmid leaving complementary sticky ends
  4. Gene combines with plasmid via complementary base pairing
  5. DNA ligase added to join sugar-phosphate backbone of gene and plasmid