recombinant DNA technology

• methods of producing fragments of DNA

  reverse transcriptase,restriction endonuclease, gene machine
• reverse transcriptase

  reverse transcriptase - joins the DNA nucleotides with the mRNA sequence

  • creates cDNA (complementary DNA)

  DNA polymerase - creates another strand using cDNA and joins it with the template strand to form a complete DNA fragment
• the role of reverse transcriptase in RT-PCR

  Produces (c)DNA using (m)RNA;
• restriction endonuclease

  restriction endonuclease (enzyme) - cuts DNA at staggered ends (recognition sites) to expose bases (sticky ends)

  • sticky ends used to join DNA at complementary bases
• gene machine

  identifies amino acid sequence of protein of interest

  the mRNA sequence is also identified and can find the complementary DNA sequence

  safety check using computer

  oligonucleotides created (small sections of overlapping nucleotides) - joins to DNA fragments
• promoter region

  added at the start of a DNA fragment
• terminator region

  added at the end of a DNA fragment

  • allows polymerase to detach
• use of vector:

  carries DNA fragments to host cell(plasmids)
• fragment -> vector

  restriction endonuclease cuts DNA fragment

  • inserted into a vector (plasmid) as restriction endonuclease cuts sticky ends complementary to DNA 
  • ligase joins the DNA fragment to the vector (forms a recombinant plasmid)
• two named types of enzymes used to insert DNA fragments into plasmids.

  Restriction (endonuclease/enzyme) to cut plasmid/vector;

  Ligase joins gene/DNA to plasmid/vector;
• transformation: recombinant plasmid→ host cell

  requires conditions: increase permeability allows the plasmid to pass through

  • Ca2+ 
  • heat shock
• identifying transformed cells:

  antibiotic resistance marker gene

  fluorescent markers

  enzyme markers
• fluorescent marker gene

  causes bacteria to glow 

  has GFP gene inserted to the recombinant plasmid
• antibiotic resistance marker gene:

  • inserting a gene that is resistant to antibiotics
  • bacteria without plasmids die
  • another antibiotic resistant marker gene added but adding a DNA fragment so the gene is disrupted
  • bacteria with the plasmid dies
• in vitro cloning

  not in an organism
• how the polymerase chain reaction (PCR) is used to amplify a DNA fragment.(in vitro method)

  • Heat to 95 °C to break hydrogen bonds
  • Reduce temperature to 55 so primers bind to DNA/strands;
  • Increase temperature to 75, DNA polymerase joins nucleotides