6.1.3 (f)

Question 1

What are the basic principles of genetic engineering?

Answer 1

• Extracting the desired gene (based on a desired characteristic) and placing in another organism using a vector
• Producing recombinant DNA: DNA from one organism that contains a gene from another organism
  
  • Giving a transgenic organism: An organism with recombinant DNA

Question 2

Describe the process of isolating the desired gene

Using restriction enzyme

Answer 2

• Restriction endonuclease enzymes cut the desired gene out of the chromosome as its active site is specific to a receptor site
• Some make clean cuts – blunt ends, but many cut the strands unevenly leaving one strand longer than the other, so there are exposed bases – sticky ends
• Sticky ends make it easier to insert the desired gene

Question 3

Describe the process of isolating the desired gene

By extracting mRNA

Answer 3

Another method uses extracting mRNA

• Extract the mRNA from the cell and use the enzyme reverse transcriptase to produce a single strand of complementary DNA (cDNA) (giving us the desired gene)
• Advantage: Easier to identify the desired gene and some cells make specific types of mRNA – such as beta cells for insulin

Question 4

When forming recombinant DNA, what vector is used and why?

Answer 4

• The most common vector used is plasmid – circular DNA molecules separate from chromosomal DNA so they can replicate independently
  
  • Once a plasmid is in a host cell it can combine with the host DNA and form recombinant DNA
• The plasmids that are chosen have two genetic markers

Question 5

Why are genetic markers used in plasmids (vectors)?

Answer 5

• They have 2 added characteristics allowing scientists to identify if the bacteria has taken up the plasmid
• E.g. It has a fluorescence gene and is resistant to penicillin
• The desired gene is placed within the fluorescent gene, and plasmid placed in bacteria
• The bacteria are cultured, if the bacteria is resistant to penicillin but not fluorescent, the bacteria has taken up the plasmid (successfully engineered)

Question 6

Describe the method for transferring the vector to the host cell:

Using restriction enzyme and DNA ligase

Answer 6

• To insert the DNA fragment into a plasmid, first cut the plasmid open
  
  • Using the same restriction endonuclease used to extract the desired gene to ensure complementary sticky ends
  • Once the complementary bases of the 2 sticky ends line up, DNA ligase forms the phosphodiester bonds between sugar and phosphate of the 2 DNA strands to join them
• The desired gene is usually placed in one of the genetic markers

Question 7

Describe the method for transferring the vector to the host cell:

Electroporation

Electrofusion

Answer 7

Electroporation: Apply an electrical current to the bacteria, making the membranes porous so the plasmids enter the cell

• The new DNA then passes through the nuclear membrane and fuses with nuclear DNA
• The electrical current must be controlled, or the membrane will be permanently damaged destroying the cell

Electrofusion: Apply an electric current to the membranes of 2 cells causing them to fuse (making 1 cell), the plasmid can then fuse with the host cell DNA

Question 8

What other method can be used to transfer the plasmid to the host cell?

Answer 8

• Culture the bacterial cells and plasmids in a calcium-rich solution and increase the temperature
  
  • The bacterial membrane is more permeable, and the plasmids enter