Recombinant DNA technology

• How is it possible to produce transgenic organisms?

  Recombinant DNA technology produces transgenic (genetically modified) organisms because:

  • The genetic code is universal - the same triplets code for the same amino acids in all organisms
  • Transcription and translation are also universal processes
• Name the 6 stages to recombinant DNA technology

  1. Isolating a gene
  2. Amplification of a gene to produce many copies
  3. Insertion of gene into a vector
  4. Transformation of cells with the vector
  5. Identification of transformed cells
  6. Culturing of cells which have taken up the vector
• What are the three ways to isolate a gene?

  1. Reverse transcriptase
  2. Restriction endonucleases
  3. Gene machine
• Describe isolating a gene using reverse transcriptase

  1. Find cells expressing the mRNA in large amounts and extract them
  2. Mix the mRNAs with reverse transcriptase to convert them to DNA without introns
  3. DNA polymerase is used to produce double-stranded copy of the gene
• Describe isolating a gene using restriction endonucleases

  REs are enzymes that cut DNA at palindromic recognition sites. Some REs leave sticky ends (exposed bases) and some leave blunt ends

  1. One RE cuts at a recognition site at the start of the gene, and a different RE cuts at a recognition site at the end of the gene
  2. This removes the double-stranded gene from the DNA
  3. The introns are then removed from the gene
• Describe isolating a gene using a gene machine

  Contains a store of DNA nucleotides and a computer that can assemble them in a specific order

  1. The gene's base sequence is typed in without introns, and the nucleotides are assembled in the correct order
  2. DNA polymerase is used to produce a double-strabded copy of the gene
• Describe stage 2

  Done using polymerase chain reaction (PCR) which is an in vitro (outside cells) technique to make lots of copies of DNA
• Outline the PCR process

  1. DNA is heated to 95 degrees which breaks the hydrogen bonds between the bases, separating the strands
  2. Then cooled to 55 degrees. This allows primers to anneal to the end of each strand
  3. Free DNA nucleotides are also attracted to their complementary bases
  4. Then heated to 72 degrees, the optimum temperature for thermostable DNA polymerase to bind the primers and form phosphodiester bonds between nucleotides
  5. The cycle is repeated to produce millions of copies of the original DNA
• Describe the importance of primers

  1. They allow DNA polymerase to bind to the end of each strand and start joining nucleotides
  2. They're made to be complementary to the ends of strands so that only target DNA is amplified
  3. They stop strands from joining back together
• Describe a vector

  • A vector is a molecule that transfers a gene into the cell
• Describe modifying the gene before stage 3

  • add promoter region to allow RNA polymerase to bind
  • add terminator gene to allow RNA polymerase to detach
• Describe stage 3 - the insertion process

  1. The gene and vector are cut with the same restriction endonucleases, leaving complementary sticky ends
  2. DNA ligase forms phosphodiester bonds between nucleotides to splice the gene and plasmid together
• Describe the 3 outcomes of insertion

  1. A recombinant plasmid - target gene inserted
  2. A non-recombinant plasmid - no target gene inserted
  3. Two target genes stick together
• Describe transforming cells with the vector

  1. The plasmids and bacterial cells are mixed with calcium ions and exposed to heat shock
  2. The cell surface membranes of the bacterial cells become more permeable, allowing plasmids to pass through the cytoplasm
• Describe identification of the cells that have been transformed

  • Only 1% of bacteria are ever successfully transformed with a plasmid
  • Of the 1% of transformed cells, some won't have the recombinant plasmid

  Marker genes:

  • A gene found on the plasmid that produces a protein which causes a visual effect
  • Marker genes can be found naturally on the plasmid or artificially inserted
• Name stage 6

  Cells with the recombinant plasmid are grown in large quantities