Gene Technology Flashcards
What is recombinant DNA
DNA that has been formed artificially by combining DNA from different organisms
What are the 3 ways of isolating a gene of interest
Reverse transcriptase
Restriction endonuclease
The gene machine
Reverse transcription
This enzyme makes DNA copies from mRNA and it naturally occurs in viruses such as HIV
A cell that naturally produces the protein of interest is selected
These cells should have large amounts of mRNA for the protein
Reverse transcriptase joins the DNA nucleotides with complementary bases to the mRNA sequence
Single stranded DNA is made (cDNA)
DNA polymerase is used to make this double stranded
Restriction endonucleases
Cut plasmid in host organism and other gene with same endonuclease
This produces complementary sticky ends as they will be a palindromic sequence
DNA ligase then joins the phosphate backbone by creating a phosphodiester bond
The gene machine
DNA fragment is created in the lab using a computer
Scientists examine protein of interest to find the AA (amino acid) sequence
Can then work out the mRNA sequence from the DNA sequence
This is entered into the computer
Oligonucleotides are then created and stuck together to produce the DNA fragments
What are the 5 stages of genetic engineering
Isolating
Inserting
Transformation
Identification
Growth
What is the most common vector for genetic engineering
Plasmid
What is transformation
Placing the vector back into the host cell - the host cell is said to be ‘transformed’
What are 5 ways of transformation
Heat shock
Gene guns
Using viruses
Liposome wrapping
Microinjection
Heat shock
Place host cells in ice cold calcium chloride solution to make cell walls more permeable
Plasmid is then added and the mixture is heat shocked (42 degrees for 2 minutes) which encourages the cells to take up the plasmids
Gene guns
DNA is shot into the cell at high speed carried on minute gold or tungsten pellets
Using viruses
Virus can be engineered to carry the desired gene and then infect the animals cells carrying the DNA with it
Liposome wrapping
Gene is wrapped in liposomes which fuse to the cell membrane and can pass through it to deliver the DNA into the cytoplasm
Microinjection
DNA is injected into a cell into a very fine micropipette
This is manipulated using a micromanipulator in order to not destroy the cell
How do we check if a cell has been transformed?
Marker genes can be inserted into the vector at the same time as the DNA fragment e.g. antibiotic resistance of fluorescence which will show up once cultured
Why do we use agrobacterium tumefaciens in GM plants
It causes tumours (crown galls) in plants so we know where the affected cells are
What is the Ti plasmid
A plasmid in agrobacterium tumefaciens
How do we make more GM plants
Take plant cells from crown galls and grow by tissue culture. Transfer tissue culture to another gel to develop roots and shoots. Clone plants
GM soya beans
2 reasons
Resistant to herbicides (roundup ready) - Soybeans can be outcompeted by weeds so farmers spray herbicides (which also kill soybean plants)
Fatty acid balance - soya beans contain lots of linoleum acid (bad for us and make oil go off) so are modified to contain more oleic and stearic acid as it’s healthier and oil lasts longer
What is a knockout mouse
A laboratory mouse in which one or more genes have been turned off or “knocked out”
Why do we use knockout mice
To create an animal model for the disease making it easier to create cures
What is the debate around knockout mice
It is considered unethical to manipulate and use mice for scientific research
Without this our understanding of diseases and development of treatments would take much longer
What is Gene cell therapy
Inserting a healthy gene into a human to cure disease of a faulty one e.g. cystic fibrosis
Process of gene therapy
Isolate the healthy allele (target gene)
Healthy allele is inserted into a retrovirus (vector) as they replicate and copy their DNA into host cells
Inject the vector (retrovirus) into the blood
Use a control injection (inject retrovirus without gene)
Monitor the progression of the disease
Compare with a control e.g. untreated organism
Need to repeat the treatment and replicate the investigation for validity
