module 6 - 21.4 genetic engineering Flashcards
what is genetic engineering?
manipulating an organisms genome
what are the basic principles of genetic engineering?
isolating a gene for a desirable characteristic in one organism and placing it into another organism
what is ‘transgenic’?
an organism that carries a gene from another organism (also referred to as a genetically modified organism - GMO)
what is the first stage of genetic modification?
isolate desirable gene
what is the process involved in isolating a gene using the enzyme restriction endonucleases?
- most common technique uses enzymes called restriction endonucleases, to cut required gene from the DNA of an organism
- these enzymes cut the DNA strands unevenly, resulting in exposed bases called sticky ends
- stick ends make it easier to indert desired gene into DNA of different organism
what is the process involved in isolating a gene using the enzyme reverse transcriptase?
- used to produce a single strand of complementary DNA
- advantageous because makes it easier to identify desired gene as particular cell will make some very specific types of mRNA
what is the formation of recombinant DNA?
DNA isolated by restriction endonucleases must be inserted into a vector that can carry it into a host cell
what are the most commonly used vectors in genetic engineering?
bacterial plasmids - small circular molecules of DNA separate from chromosomal DNA that can replicate independently
why are bacterial plasmids used in genetic engineering?
- once plasmid gets into new host cell it can combine with host DNA to form RECOMBINANT DNA
- plasmids contain marker gene
what is a marker gene?
allows scientists to determine that the bacteria have taken up the plasmid by growing bacteria in media containing the antibiotic
how do you insert DNA into a plasmid?
- DNA fragment must be cut open using same restriction endonuclease used to isolate the DNA
- results in plasmid having complementary sticky ends to sticky ends of DNA fragment
- once sticky ends are lined up, DNA ligase forms phosphodiester bonds between sugar and phosphate groups on the 2 strands of DNA, joining them together
why might plasmids be given a second marker gene?
- used to show that the plasmid contains the recombinant gene
- it placed in plasmid by genetic engineering methods
- plasmid then cut by restriction enzyme within marker gene to insert desired gene
- if insertion is successful, marker gene will not function
how do you transfer the vector?
plasmid with recombinant DNA must be transferred into host cell through process called transformation
what are the 2 methods of transferring a vactor?
- culture bacterial cells and plasmids in calcium rich solution
- electroporation
what is involved in the method of culturing bacterial cells and plasmids in calcium-rich solution?
- culture bacterial cells in calcium-rich solution and increase temperature
- this causes bacterial membranes to become permeable and plasmids can enter
what is involved in electroporation?
- small electrical current is applied to bacteria
- this makes membranes very porous so plasmids move into cells
- can be used to get DNA fragments directly into eukaryotic cells, new DNA will pass through cell membrane and nuclear membrane to fuse with the nuclear DNA
- technique’s less useful in whole organisms
what is electrofusion?
- tiny electric currents are applied to the membranes of 2 different cells
- this fuses the cell and nuclear membranes of 2 cells together to form a hybrid/polyploid cell, containing DNA from both
- important in production of nonclonal antibodies
how are monoclonal antibodies produced?
produced by combination of cell producing one single type of antibody with a tumour cell, meaning it rapidly divides in culture
What is one method of genetically modifying plants?
- Using agrobacterium tumefaciens (a bacterium that causes tumours in healthy plants)
- Desired gene is placed in the plasmid of A. tumefaciens along with maker gene
- this is then carried directly carried into me plant cell DNA
- transgenic plant cells form a callus due to bacteria plasmid
What is a callus?
Mass of GM plant cells, each which can be grown into a new transgenic plant
How can transgenic plants be produced by electrofusion (as well as using A. Tumefaciens)?
- The cells produced have chromosomes from both original cells so are polyploid
- The cells that are fused may be from similar species or very different ones
Main stages in this process:
1. Removal of cell wall by cellulases
2. Electrofusion to form a new polyploid cell
3. Use of plant names to stimulate growth of new cell wall
4. Callus formation and production of many cloned, transgenic plants
Why is it harder to engineer the DNA of eukaryotic animals, tran bacteria or plants?
Animal cell membranes are less easy to manipulate
What is the summarised whole process of genetic engineering in plants?
- Cut plant
- Expose leat to bacteria carrying a resistant die and an antibiotic resistant gene. Allow bacteria to deliver the genes into leat cells
- Expose leaf to antibiotic to kill plus hat lack new genes, wait for surviving (gene-altered) cells to multiply and form a clump (callus)
- The plants are transferred to soil to develop into fully differentiated adult plants that are resistant
