Manipulating Genomes : Genetic Engineering Flashcards
Genetic engineering and what makes this possible?
Manipulation of the DNA sequences of an organism
- genetic code is UNIVERSAL - nearly all use same 4 bases + same codon code for same amino acids in all living things
What is recombinant DNA , transgenic organism, and genetically modified organisms?
RECOMBINANT DNA: altered DNA, with the introduced nucleotides
TRANSGENIC ORGANISM: organism that contains nucleotide sequences from different species
GENETICALLY MODIFIED ORGANISM (GMO): any organism that has introduced genetic material
Uses of genetic engineering?
Genetic modification of crops - increase YIELDS
- resistance to drought,disease,pesticide and herbicides /provide increased nutritional value
Genetic modification of livestock - disease/pest resistance and increased productivity
Genetic modification of bacteria - produce medicines (insulin ), decompose toxic pollutants, carry out large scale chemical production
Enzymes used in genetic engineering techniques?
RESTRICTION ENDONUCLEASES: cut genes/plasmids at specific base sequences (restriction sites).
LIGASE - joins together the cut ends of DNA by forming phosphodiester bonds - gene and plasmid joined tgt
REVERSE TRANSCRIPTASE - Used to build single stranded DNA from single stranded RNA
What are vectors and the 3 types?
Vectors - used to deliver DNA fragments into a cell
PLASMIDS: transfer DNA into bacteria/yeast
VIRUSES : transfer DNA into human cells/bacteria
E.g bacteriophage infect bacterium by injecting its DNA into it/integrates with bacterial DNA
LIPOSOMES : fuse will cell membranes to transfer DNA into cells
What are markers and the 3 types?
Markers - genes that code for identifiable substances that can be tracked
Fluorescent markers e.g. green fluorescent protein (GFP) which fluoresces under UV light
Enzyme markers e.g. β-glucuronidase (GUS) enzyme which transforms colourless or non-fluorescent substrates into products that are coloured or fluorescent
Antibiotic resistance marker genes - include antibiotic resistance gene, as well as desired gene in vector
Plate bacteria onto medium containing antibiotic
Those that survive have antibiotic resistance gene /have been genetically modified
Uses of genetic engineering?
GM microorganisms: make recombinant proteins - research/treatment (cancer,diabetes)
GM plants/animals - meet global food demands/produce protein for meds
GM Crops - resistance to herbicides/pests/enriched in vitamins
- reduce impact of farming on environment due to less need for pesticides
GM livestock
GM salmon
GM pathogens
Advantages of genetic engineering microorganisms to produce recombinant human proteins ?
- cost effective to produce large volumes
- simpler
- faster to produce many proteins
- reliable supply
- proteins are engineered to be identical to human proteins or have beneficial modifications
- can solve the issue for people who have moral or ethical or religious concerns against using cow or pork produced proteins
Why are recombinant human proteins produced using eurkaryotic cells?
These cells carry out POST TRANSLATIONAL MODIFICATION (due to presence of Golgi apparatus/enzymes) - needed to produce suitable human protein
benefits of using genetic engineering rather than selective breeding techniques to solve the global demand for food?
Organisms with the desired characteristics are produced more quickly
All organisms will contain the desired characteristic (there is no chance that recessive allele may arise in the population)
The desired characteristic may come from a different species/kingdom
How is livestock modified?
- Pharming - produce pharmaceutical drugs
- biopharm sheep/goats genetically modified to produce many useful human proteins imm their milk
How are GM salmon (AquaAdventure salmon) genetically modified ?
- genetically modified to GROW RAPIDLY due to growth hormone being produced throughout year, instead of just spring/summer
- has a product to sell in half the time —> INCREASED YIELD
- combined growth hormone gene from chinook salmon with promoter gene from ocean pout —> ensures gene is continually expressed
- alls salmon are female/sterile - prevent reproducing in wild
How are pathogens genetically modified ?
- modified to shed light on their metabolism, drug resistance / how it causes damage to its host
- development of effective vaccines and drugs can be aided by this research
Adenoviruses can be genetically altered to act as vectors in gene therapy - ideal vectors as they are not cell-specific or species-specific - infect cells of many mammals
Specific genes are removed from the virus so that it can not replicate inside host cells, creating space for the insertion of other desired genes
Method of genetic engineering: How is human insulin produced?
- Bacteria plasmids modified to include HUMAN INSULIN GENE
- ISOLATION OF DESIRED GENE: in this case, mRNA from beta cells of islets of langerhans used + add reverse transcriptase to form single stranded cDNA and form double strand using DNA polymerase (addition of free nucleotides at the ends of DNA form sticky ends)
DNA ligase is used to splice the plasmid and human DNA together - recombinant plasmids are mixed with E.Coli and then inserted into E.coli by transformation (host cells are subject to heat shock in presence of calcium chloride —> increase permeability of host cells)
(Electroporation can be used instead for other genetic engineering processes) - the transgenic bacteria are identified (by the markers), they are isolated, purified and placed into fermenters that provide optimal conditions
- The transgenic bacteria multiply by binary fission, and express the human protein - insulin, which is eventually extracted and purified
How has genetic enrgineering led to insect resistance in a type of soya?
genetically modified the already herbicide-resistant variety of soybean (Roundup Ready™, RR1) by inserting a gene for the Bt toxin
- plants with Bt toxin gene produce own insecticides
- however insect have developed resistance to genes for Bt toxin —> less effective
How is TMV used a vector?
- gene for hormone TMOF inserted into cells of crops via GM tobacco mosaic virus
- modified TMV sprayed on surface of crops where it can invade plant cells
- host plant cells TRANSCRIBE THE GENE to produce hormone TMOF - inhibit production of trypsin within insect pests
- leaves of GM crop exposed to GM virus collected/ground into powder to create REPELLENT SPRAY again mosquitoes
Arguments against use of GMOs?
- companies charge farmers more money for GM seeds
- object to use of GMO’s in food production due to lack of long term research on effects on human health
- without appropriate labelling consumer cant make informed decision abt consumption of GM food/choices being made for them
- pollen from GM crops contaminate non GM crops that are organic
- reduce biodiversity
- herbicide resistance genes transfer to weed plants - ‘superweeds’
What is gene therapy and why are most in clinical trail stage?
Involves using various mechanisms to alter a person’s genetic material to treat/cure diseases
- scientist find it difficult finding delivery systems to transfer normal alleles in person’s cells /ensure gene is correctly expressed once there
What vector are used as a delivery system in gene therapies?
Viruses : have mechanisms needed to recognise cells/deliver genetic material into them
Non viral vectors (liposomes/ ‘naked’ DNA)
2 types of somatic gene therapy? Why is the genetuc material not inherited by future generations?
EX VIVO: new gene inserted via a virus vector into the cell outside the body
- Blood/ bone marrow cells extracted and exposed to the virus which inserts the gene into these cells - cells are then grown in the lab and returned to the person by injection
IN VIVO – the new gene is inserted via a vector into cells inside the body
Changes in genetic material are targeted to specific cells /doesn’t target gametes
Why is inserting genetic material into germ cells illegal?
- germ cels involved in sexual reproduction
- changes made to genetic material is potentially permanent /can be inherited
How is Severe Combined Immunodeficiency (SCID) treated?
caused by the body’s inability to produce adenosine deaminase (ADA), an enzyme for functioning of the immune system
- EX VIVO SOMATIC GENE THERAPY
- virus transfers normal allele of ADA into T lymphocytes removed from patient
- cells returned via injection
- not permanent cure - T lymphocytes are replaced by body over time so need REGULAR TRANSFUSIONS every 3-5 months
Why are other viruses not used as vectors in gene therapy for SCID?
Retroviruses : viruses insert their genes randomly into a host’s genome - they could insert the gene into another gene or into a regulatory sequence of a gene (which could result in cancer)
- caused Leukaemia
Why are lentiviruses/adeno-associaed viruses used as vectors for SCID now?
LENTIVIRUSES: randomly insert their genes into the host genome but can be modified to not replicate
ADENO-ASSOCIATED VIRUSES don’t insert their genes into the host genome and therefore the genes are not passed onto the daughter cells when a cell divides.
How are inherited eye diseased treated?
E.g Leber Congenital Amaurosis - cause blindness
in vivo somatic gene therapy: doctors injected the retina adeno-associated viruses - contained the normal alleles of one of the genes that caused damage to the photoreceptors
What is electroporation?
When an electrical current is applied to the membrane to make it more porous
3 ways to isolate desired gene for genetic engineering/modification?
Restriction endonucleases: cut genes/plasmids at specific base sequences (restriction sites). Different restriction enzymes cut at different restriction site - cut across double strands at 2 places (place where they cut is a STICKY END)’
Reverse transcriptase: enzyme converts RNA to DNA
- found in RETROVIRUSES - which stores genetic info in form of RNA
Synthesising gene in vitro using POLYNUCLEOTIDE SYNTHESISER - if nucleotide sequence of gene is known, this sequence can be fed into computer
Process of using reverse transcriptase to isolate desired gene?
- Cells that express desired genes produce many transcripts of mRNA
- MRNA transcripts used as template for synthesis of single stranded complementary DNA (cDNA)
- MRNA template hydrolyses with enzyme to isolate single stand of cDNA
- Double stranded DNA made by DNA polymerase using cDNA a template
Double stranded DNA has ‘instructions’ needed to code for desired protein
Why is using reverse transcriptase rather than restriction endonucelases better?
Introns have already been spliced out
- so gene can be correctly transcribed/translated by prokaryotic cells
Introns - sections of DNA/RNA that doesn’t code for any proteins
How can polynucleotide sequencer be used to isolate desired genes?
- known nucleotide sequence fed into computer
Oligonucleotides synthesised by polynucleotide synthesiser in AUTOMATED PROCESS - short sequences of nucleotides that overlap with each other
Gene is then assembled by joining tgt overlapping oligonucleotides / amplified using PCR
Why are there few ethical concerns with genetic engineering of E.Coli?
- Few animal rights to consider
- safety of genetic engineering has been established
Explain how inserting a new gene into a chromosome could affect the functioning of other genes in that chromosome?
Altered triplets
- adjacent genes will be switched on/off
Use of electroporation?
Increase rate at which bacteria take up recombinant plasmids
Purpose of sticky ends when producing recombinant DNA?
Join 2 pieces of DNA together
By complementary base pairing
Suggest how a herbicide resistance gene can be used a genetic marker (3)
Insert herbicide resistance gene with the desired gene
Apply herbicide to transformed plant cells
Survivors have the desired gene (as they’re herbicide resistant)
