genetic engineering

Question 1

genetic engineering definition

Answer 1

the manipulation of a genome

Question 2

stages of genetic engineering

Answer 2

• isolating the desired gene
• the formation of recombinant DNA
• transformation
• identification
• growth

Question 3

techniques of isolating the desired gene

Answer 3

• using restriction endonucleases
• isolating mRNA for the desired gene and using reverse transcriptase

Question 4

transgenic

Answer 4

• an organism that carries a gene from another organism
• often also called genetically modified organism (GMO)

Question 5

isolating the desired gene using restriction endonucleases

Answer 5

• each type of endonuclease is restricted to breaking the DNA strands at specific base sequences within the molecule
• some make clean, blunt end cut in the DNA
• many restriction endonucleases cut the 2 DNA strands unevenly, leaving one of the strands of the DNA fragment a few bases longer than the other (sticky end)
• make it easier to insert the desired gene into the DNA of a different organism

Question 6

isolating the desired gene using mRNA and reverse transcriptase

Answer 6

• isolating mRNA for the desired gene
• using reverse transcriptase to produce a single strand of complementary DNA (cDNA)
• plasmid and cDNA fused using DNA ligase
• recombinant plasmid introduced to host cells
• bacteria multiply in a fermenter and produce insulin
• separation and purification of human insulin
• human insulin can be used by diabetic patients

Question 7

advantages of isolating desired gene using mRNA and reverse transcriptase

Answer 7

• makes it easier to identify the desired gene
• as a particular cell will make some very specific types of mRNA
• e.g. Beta cells in pancreas make insulin so produce lots of insulin mRNA molecules

Question 8

what are the most commonly used vectors in genetic engineering

Answer 8

bacterial plasmids (small circular molecules of DNA separate from chromosomal DNA that can replicate independently)

Question 9

recombinant DNA

Answer 9

formed by host DNA combined with plasmid which just entered the host cell

Question 10

how do scientists know if the bacteria has taken up the plasmid

Answer 10

• specific plasmids are chosen as vectors because they contain a marker gene
• they may have been engineered to have gene for antibiotic resistance
• enables scientists to determine that the bacteria have taken up the plasmid by growing the bacteria in media containing the antibiotic

Question 11

how to insert a DNA fragment into a plasmid

Answer 11

• same restriction endonuclease as used to isolate the DNA fragment is used to cut the plasmid
• so the plasmid has complementary sticky ends to the DNA fragment
• once the complementary bases of the 2 sticky ends are lined up, DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the 2 DNA strands, joining them

Question 12

how do scientists know that the plasmid took up the recombinant gene

Answer 12

• vector plasmid is given a second marker gene
• marker gene is placed in the plasmid by genetic engineering methods
• plasmid is cut by restriction endonuclease within this marker gene to insert the desired gene
• if the DNA fragment is inserted successfully, the marker gene will not function

Question 13

development of using different types of marker genes

Answer 13

• concerns about antibiotic resistance in genetically engineered organisms
• so genes producing fluorescence or enzyme causing colour change in a particular medium are now more widely used as marker genes
• if a bacterium does not fluoresce, or change colour of the medium, it has been engineered successfully and can be grown on

Question 14

what happens in transformation

Answer 14

• plasmid with recombinant DNA must be transferred into host cell
• either with electroporation, using calcium-rich solution, or electrofusion

Question 15

transferring vector via calcium rich solution

Answer 15

• culture the bacterial cells and plasmids in a calcium-rich solution and increase the temp
• causes bacterial membrane to become permeable and plasmids can enter

Question 16

transferring the vector via electroporation

Answer 16

• small electrical current is applied to the bacteria
• makes membranes very porous and so plasmids move into the cells
• DNA fragments can also be moved directly into eukaryotic cells
• new DNA will pass through the cell membrane and nuclear membrane to fuse with the nuclear DNA
• very effective technique but power of electrical current has to be carefully controlled or the membrane gets permanently damaged/destroyed
• less useful in whole organisms

Question 17

electrofusion for plant cells

Answer 17

• tiny electric currents are applied to the membranes of 2 different cells
• fuses cell and nuclear membranes of 2 different cells together to form hybrid or polyploid cell, containing DNA from both
• successful for GM plants

Question 18

electrofusion for animal cells

Answer 18

• animal cells dont fuse as easily/effectively as plant cells as their membranes have different properties
• polyploid animal cells (especially mammalian) dont usually survive in body of living organism
• but electrofusion is important in production of monoclonal antibodies, produced by a combination of a cell producing one single type of antibody with a tumour cell
• so it divides rapidly in culture
• monoclonal antibodies are now used to identify pathogens in both animals and plants and for treatments of diseases e.g. some forms of cancer

Question 19

engineering prokaryotes

Answer 19

• bacteria/other microorganisms have been genetically modified to produce many different substances that are useful to people
• including hormones e.g. insulin, human growth hormone, clotting factors for haemophiliacs, antibiotics, pure vaccines, many other enzymes used in industry

Question 20

engineering plants

Answer 20

• one method used Agrobacterium tumefaciens which causes tumours in healthy plants
• a desired gene (e.g. herbicide resistance) is placed in the Ti plasmid of A. tumefaciens along with a marker gene e.g. antibiotic resistance/fluorescence
• this is carried directly into the plant cell DNA
• the transgenic plant cells form a callus (mass of GM plant cells which can each be grown into a new transgenic plant)
• or electrofusion produces transgenic plants also
• cells produced have chromosomes from both of the og cells and so are polyploid
• cells that are fused may be from similar species or very different ones
• main stages of this process involve removal of the plant cell wall by cellulases, electrofusion to form a new polyploid cell, use of plant hormones to stimulate growth of a new cell wall, followed by callus formation and production of many cloned, transgenic plants

Question 21

engineering animals

Answer 21

• much harder to engineer DNA of eukaryotic animals (especially mammals) than bacteria/plants
• because animal cell membranes are less easy to manipulate
• but it is an important technique both to enable animals to produce some medically important proteins and to try cure human genetic diseases e.g. CF and Huntington’s disease