Recombinant DNA Technology

• What is recombinant DNA technology?

  Transfer of DNA fragments from one organism or species, to another
• Explain why transferred DNA can be translated within cells of recipient

  (transgenic) organisms:

  1)Genetic code is universal

  2) Transcription and translation mechanisms are universal
• Describe how DNA fragments can be produced

  using restriction enzymes:

  1)Restriction Endonucleases cut/digest DNA at specific base ‘recognition

  sequences’ either side of the desired gene

  ○ Shape of recognition site complementary to active site

  2. Many cut in a staggered fashion forming ‘sticky ends’

  (single stranded overhang)
• Describe how DNA fragments can be produced from mRNA:

  1)Isolate mRNA from a cell that readily synthesises the protein coded for by the desired gene

  2) Mix mRNA with DNA nucleotides and reverse transcriptase → reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)

  3) DNA polymerase can form a second strand of DNA using cDNA as a template which creates dsDNA
• Suggest two advantages of obtaining genes from mRNA rather than directly from the DNA removed from cells:

  ● Much more mRNA in cells making the protein than DNA → easily extracted

  ● In mRNA, introns have been removed by splicing (in eukaryotes) whereas DNA contains introns

  ○ So can be transcribed & translated by prokaryotes who can’t remove introns by splicing
• Describe how fragments of DNA can be produced using a gene machine:

  ● Synthesises fragments of DNA quickly & accurately from scratch without need for a DNA template

  ○ Amino acid sequence of protein determined, allowing base sequence to be established

  ● These do not contain introns so can be transcribed & translated by prokaryotes