Gene Technology Flashcards
What are GMO’s
Organisms with recombinant DNA
Can be split into genetically engineered microorganisms (GEM’s) and others
What are the containment methods for GEM’s
⭕️ bacterial strains ill-adapted to the human physiology - minimum temp tolerance above human body temp
⭕️ contain ‘suicide genes’ - activated if move outside certain pH or temp range
⭕️ purpose-built licensed laboratories
⭕️ tightly controlled procedures for staff - cleaning etc
What are the three methods of genetically modifying a plant
- Use a tumour inducing plasmid from the common soil bacterium
- Use gene guns
- Use plant viruses
Describe how you would use a tumour inducing plasmid to genetically modify a plant
- Agrobacterium tumefaciens invades damaged plant tissue
- tumour-inducing (Ti) plasmid transferred to plant cell
- desired gene spliced into plasmid
- readily taken up by plant cell provided cell wall has been removed by enzyme cellulase
Explain how gene guns are used to genetically modify a plant
- minute pellets covered in DNA w desired gene
- shot through cellulose walls
- using gene gun
What are the advantages of GM crops
See spider diagram in notes
What are the disadvantages of GM crops
See spider diagram notes
How are plant viruses used to genetically modify a plant
Infect cells by inserting their nuclei acid
Describe how to extract and cut up DNA
- restriction enzymes - cut by hydrolysis
- recognition sequence
- leaves sticky ends
Describe a sticky end
Short single stranded section of DNA
Bases are unpaired so exposed
What does reverse transcriptase do
Uses mRNA as template strand
Produces single stranded cDNA
Which is converted to double stranded DNA by DNA polymerase
What is a gene probe and what do they do
Short single strand of DNA
Binds to target gene
Confirm presence of desired gene
Identify sections of DNA
What are the steps for using a fluorescently labelled probe
- DNA cut
- Separate by gel electrophoresis
- Transferred to nylon membrane
- Treated w fluorescently labelled probe
- Probe binds
- Exposed to UV light
What is pcr
Few fragments of DNA duplicated to millions
What enzyme is used in pcr
Taq polymerase
What is the function of primers
- stop two DNA strands rejoining
- ‘bracket’ section of DNA to be copied
- DNA replication can only start with a double stranded region
What is the process of PCR
- Heat to 95°C - break H and separate
- Cooled to 50-60°C - primers annealed
- Heat to 72 °C -enzyme optimum temp
- Repeat
What is the role of PCR
Forensic analysis
Pre natal diagnosis
Historical studies
Genetic relationships
What are MRSs
- microsatellite repeat sequence
- Small no. of bases repeated many times in non-coding region
- number of repeats is unique to each individual
- probe for particular MRSs created
What are SNPs
Differences in single nucleotides
What are the steps for genetic fingerprinting
- Extract DNA
- Restriction enzymes cut out repeat sequence
- Separate fragments by gel electrophoresis
- Transfer to nylon sheet - southern blotting
- Attached labelled probe
- detect diff repeat sequence
What is the role of genetic fingerprinting
Establish paternity Forensics Tissue typing for transplants Evolutionary relationships Identify victims
What is a vector
Length of DNA
carries gene we want into host cell
What is a plasmid
Small circular ring of DNA
Bacterial cell
Copy themselves independently
What are the two methods of obtaining required gene
- Restriction endonuclease cut gene out of chromosomal DNA
2. Reverse transcriptase - produce DNA from RNA
What are the two methods for inserting gene into vector
Bacterial plasmids
Viruses
How are bacterial plasmids used to insert gene into a vector
- Plasmid cut w restriction enzyme
- Same enzyme cuts gene from donor DNA
- Gene spliced into plasmid
- Sticky ends anneal
- DNA ligase - phosphodiester bonds between sugar-phosphate backbones
- Now recombinant plasmid
How are viruses used to insert gene into a vector
- Insert genetic material into host cell
2. Transfer recombinant DNA into bacterial cell
What are the two methods for inserting the vector into a host cell
Heat shock
Electroporation
Describe the method of heat shock
- Cells incubated w recombinant plasmid
- In solution of calcium ions at 0°C
- Sudden rise in temp to 40°C
- Bacterial cells more permeable fo plasmids
Describe electroporation
- Short electrical pulse
- Temporarily disrupts membrane
- Opens pores in membrane
Describe how a marker gene is used to check cell contains recombinant DNA
⭕️ R-plasmid resistant to tetracycline and ampicillin
⭕️ restriction enzyme cuts tetracycline gene but not ampicillin
⭕️ if it has taken up original plasmid then it’s resistant to both
⭕️ if takes up recombinant plasmid then resistant to ampicillin but not tetracycline
How to use fluorescent protein to check if cell contains recombinant DNA
Gene that produces fluorescent proteins isolated and attached to gene of interest
If taken up then will glow under UV light
How to make a replica plate
- Sample on master plate
- Touch cloth on bacterial colonies on master plate
- Touch on to replica plate containing tetracycline
How to clone transformed bacteria
Cultured in fermenters
Transferred gene switched on
Optimal pH, temp and nutrients
What are the 4 methods of introducing genes into animal cells
Electroporation
Liposomes
Viruses
Microinjection
Describe how liposomes work when inserting genes into animal cells
Coat DNA in artificial lipid vesicles
Adhere to cell membrane - pass DNA into cell
What are the two types of viruses used to introduce genes into cells
Adenoviruses
Retroviruses
How do retroviruses work
RNA delivered to host
Copied to DNA
By reverse transcriptase
How do microinjections work when introducing genes to animal cells
Inject DNA directly into nucleus of fertilised egg
List some of the uses of genetically modified animals
Protein enriched milk - cow
Treatment of diseases
Treatment of skin cancer - chicken eggs
What are the two types of gene therapy
Somatic cell gene therapy
Germ line therapy
What are the pros and cons of somatic gene therapy
C - only targets affected tissue
C - only when defective gene in specific and easily reached part of the body
P - used at any stage of individuals life
What happens when functional allele is in host cell
- Donor DNA incorporated in host DNA
2. DNA functions as independent DNA unit (episome)
What is a genome
Complete nucleotide (base) sequence
What is genome sequencing
Determining order of bases in each chromosome of eukaryotic organisms
What is genetic mapping
Establishing exactly where particular genes are on chromosomes
What is a microarray
⭕️ solid silicon/glass base ⭕️ thousands of spots ⭕️ spots contain many of same DNA probe ⭕️ diff probes in diff spots ⭕️ analyse many genes simultaneously ⭕️ rapid analysis of DNA
Why use microarays
- Identify genotypes - detect defective alleles - diagnose genetic disease
- Measure gene expression - help diagnose cancer - gene more or less active
How is a microarray used
⭕️ single stranded DNA/mRNA washed over array
⭕️ complementary to probe the hybridise with it
⭕️ DNA/mRNA labelled
⭕️ diff colours = diff levels of hybridisation
How is gene expression measured using microarrays
- mRNA extracted - concerted or cDNA by reverse transcription
- Flourescent tag
- Added to array
- Complementary then will hybridise
How can a microarray be used to diagnose oncogenes
- Labelled cDNA from RNA from healthy and cancerous tissue
- Dif flourescent dyes for each
- Both added to microarray and hybridise
- Scan with laser and tags generate signals - strength of which depends on amount of cDNA binding to probes
What are the benefits of genome sequencing
- mapping of genes
- pharmacogenetics - designer drugs
- gene therapy
- DNA fingerprinting
- ancestral links
- work out primary structure of protein
What is a designer drug
- Matched to individuals profile
- more effective first time
- reduce trial and error so less suffering
- reduce side effects and allergies
What is gene knockout
Gene(s) removed or made inoperative
Allow you to determine the role of the gene
What is gene knockin
Particular gene added
Deliberately add defective gene to study the disease progression
Why are mice used in studies
Closely related to humans
Mammal
Short life cycle and easily kept in lab conditions
Most people not ethically opposed
Pros and cons of genetic screening
✅ understand risk of passing on genetic disorder
❌ stress if carrier
❌ termination of pregnancy?
