6.1.3 Manipulating Genomes Flashcards
DEFINITION- Genomics
The branch of molecular biology concerned with structure, function, evolution and mapping of genomes
DEFINITION- Gene
A section of DNA that contains the complete sequence of bases (codon) to code for a protein
DEFINITION- Thermophilic
An extremophile that thrives in relatively high temperatures
DEFINITION- Thermus Aquaticus
A species of bacteria that can tolerate high temperatures
DEFINITION- TAQ Polymerase
A thermostable DNA polymerase named after the thermophile, Thermus Aquaticus
DEFINITION- Nucleotide
The monomer unit used to for nucleic acids, composed of a pentose sugar, phosphate group and nitrogenous base.
DEFINITION- H Bond
Weak bonds that can occur whenever theres a slightly negative molecule and slightly positive H
DEFINITION- DNA Primer
A short single stranded nucleic acid utilised by all living organisms in the initiation of DNA synthesis
DEFINITION- Amplification
A mechanism leading to multiple copies of a chromosomal region of a gene.
Role of PCR
DEFINITION- Restriction enzyme
Enzymes that chop strands of DNA into small pieces,
Endonucleases
DEFINITION- DNA ligase
An enzyme involved in DNA replication, that catalyses the formation of a phosphodiester bond.
DEFINITION- Plasmid
A genetic structure in a cell that can replicate independently of chromosomes
DEFINITION- Transgenic
An organism containing genetic material into which DNA from an unrelated organism has been artificially introduced.
Restriction enzyme Details
- Extracted from bacteria
- Endonucleases
- Cut DNA at specific points to create palindromic sequences
- Leave behind sticky ends
Restriction enzyme process
- Each enzyme is specific for a certain base sequence
- The active site on the enzyme has a specific shape
- The base sequence has a complimentary shape
- DNA sugar phosphate backbone is cut at the restriction site
- Hydrolysis breaks the backbone in different places
- Leaves a staggered cut (sticky end)
Polymerase Chain Reaction steps and Role
- Denaturation
- Annealing
- Extension
Amplifies DNA
Reaction mixture PCR
Extracted DNA
Nucleotides
DNA polymerase
Primers
Denaturation PCR
95degrees
H bonds break between DNA strands
Annealing PCR
55degrees
Primers bind to strands with H bonds as DNA polymerase cannot bind to single strands
Extension PCR
72degrees
DNA polymerase adds free nucleotides in 5 to 3 direction with complimentary base pairs
Differences between PCR and DNA replication
- Only short sequences can be replicated, not whole chromosomes
- A primer is required
- Needs to heat and cool to separate the DNA strands, bind on the primers and for DNA replicate
Applications of PCR
- Forensic Science
- Detecting mutations
- Monitoring spread of disease
Advantages of PCR
- Quicker
- Requires less equipment
- Less space
- Easier and less costly to run
- Safer
Advantages to cloning in living organisms (bacterial host)
- Longer sections of DNA can be cloned
- Less prone to mutation (taq polymerase can insert wrong base)
- Less expensive set up costs
- Less technically complex
Gel Electrophoresis Details
- DNA samples are treated with restriction enzymes to create DNA fragments
- Placed into wells at the negative end of the gel
- Gel immersed into buffer solution
- Electrodes are attached so a current can pass through over a set time (2hr)
- Position of fragments can be shown using dye that stains DNA
- Separates based on size
Why DNA is drawn towards the positive pole?
Gel Electrophoresis
DNA is negative so repelled away from the -ve electrode and attracted to the positive
Why they travel different lengths
Gel Electrophoresis
Longer strands move slower because they’re heavier and theres more friction, therefore they move less distance
Which stain is used?
Gel Electrophoresis
Ethidium Bromide, binds to the DNA and shows up under UV light
Gene Probes
Short strands of DNA with complimentary base strands to the DNA being investigated, the complimentary binding is called annealing.
50-80 nucleotides long
How to label gene probes
- Radioactively with isotope phosphorus 32, identified with a photographic plate
- Fluorescently so they’ll be identified under UV light
Uses of Gene probes
- Locate a specific gene for enticing engineering
- Identify the same gene in a variety of different genomes
- Identify the presence of particular allele for a genetic disease
DNA profiling Steps
Extraction Digestion Separation More separation Hybridisation Development More Development Every Dinosaur Sings More Highly During Meetings
DNA Profiling- Extraction
DNA extracted from the sample
DNA Profiling- Digestion
Restriction endonuclease cut DNA into fragments
DNA Profiling- Separation
Fragments separated using gel electrophoresis
DNA Profiling- More Separation
DNA fragments transferred from gel to nylon membrane in a process called southern blotting
DNA Profiling- Hybridisation
DNA probes are added to label the fragments, the radioactive probes attach to specific fragments.
DNA Profiling- Development
Membrane with radioactively labelled DNA fragments is placed onto an Xray film
DNA Profiling- More Development
Development of an Xray film reveals dark bonds where radioactive Fluro DNA Probes have attached.
Benefits of using Genomes for medicines
- More powerful medicines
- Better and safer first time
- Decrease in overall cost of healthcare
Gene Sequencing Reaction Mixture
-DNA polymerase
-Many copies of single stranded DNA template
-Free DNA nucleotides
-Modified DNA nucleotides with fluorescent marker
-Primers, approx 20 Base pairs
(All inside a Thermal Cycler)
Gene Sequencing Description
- Primer attaches to 3’ end of template strand allowing DNA polymerase to attach (Annealing) 55degrees
- Free nucleotides are added forming C Base pairs (Extension) 72degrees
- Polymerase enzyme thrown off when modified nucleotide attaches, acts as a terminator base, each base a different colour. (Extension interrupted)
- Varying lengths of DNA strands can be ordered with the end nucleotide tagged with specific colour
- Pulled through electrophoresis and the colours are read by lasers
Next generation Sequencing
- Easier and quicker
- Uses plastic slide instead of gel
- Still uses coloured terminator bases
Uses for Comparing Genomes
- Identify genes that are essential for life
- Verifies useful genes are conserved in evolution
- Help show evolutionary distance
- Predicting amino acid sequences
Genetic engineering
Genetic material can be changed, the gene from one species organism can be inserted into another organism
Reasons to genetically Engineer
- Improve features of an organism
- Resistance to herbicides
- Promote muscle growth - Allow organisms to synthesise useful product
- Insert insulin gene
- Golden rice
- Female sheep can produce proteins in milk
Overview of Genetic Engineering
- Obtaining gene
- Gene is inserted into a vector
- Vector inserts gene into cell
- Identifying transformed cells
Obtaining a Gene
Once identified, it can be cut out a chromosome using restriction enzymes or made via reverse transcription of mRNA. Genes can be sequenced using an automated polynucleotide sequencer.
Inserting genes into vectors
e. g. Bacterial Plasmids
- Plasmid must be cut with the same restriction enzymes to create complementary sticky ends.
- Plasmid and gene are then mixed with DNA ligase and some combine to form recombinant plasmids.
- Ca Salts and Heat shock can help plasmids enter the bacteria, only 1% do
- Transformed bacteria contain new DNA and are therefore Transgenic.
Vector inserts gene into cells
- Viral Transfer, vectors in a virus and the cells are infected inserting DNA in directly.
- Ti Plasmids, tumour inducing for plants
- Liposomes, DNA is wrapped in lipid molecules they’re fat soluble so pass through the membrane
- Direct method DNA shot into cell with mini gun
Identifying Transformed Cells
- Fluorescent Marker Genes, found in jellyfish and can be inserted into plasmids with genes so if they glow they have taken up the plasmid
- Antibiotic Marker Genes, plasmids often contain 2 antibiotic resistant genes, if plasmid is then up they lose one, then use replica plating
Replica Plating
- Master plate contains standard nutrient agar and all bacteria colonies grow.
- Block covered in sterile velvet is placed on it and bacteria sticks
- Block is then placed on agar containing 1st antibiotic
- Only bacteria with plasmid grow
- Then stamped on 2nd antibiotic and only bacteria with resistance to 2nd antibiotic grows
- Use to identify bacteria to be grown on a large scale
Gene Therapy
Using genetic technology to treat genetic disorders, replace dysfunctional genes with working copies into cells.
Somatic Cell Therapy
Adding genes- replace faulty gene
Killing specific cells- programme cancerous cells to express genes that make them weak to the immune system
Germ line Therapy
Embryo cells have the potential to specialise into any cell type, if a gene is placed in these every body cell will possess it.
However is unethical and illegal
Somatic Cell Gene Therapy Issues
- Tricky to get genes into specific cells
- Short lived so must be repeated
- Only affects the actual patient
- Difficult to get gene to function
Germline Gene Therapy Issues
- More straightforward to deliver gene into cell
- All subsequent cells have the functioning gene
- Unethical to engineer human embryos
- Genetic manipulation is passed onto children
Problems with Vectors
- Immune response, attacks foreign molecules
- Inefficient, liposome take up rate is low
- Side Effects, may cause tumours
- Viral vectors may cause disease
Gene Therapy- Cystic Fibrosis
- Gene works incorrectly so sticky mucus builds up
- some trials using liposomes
Gene Therapy- SCID
- Severe Combined Immunodeficiency disease
- T lymphocytes are destroyed due to the build of of toxins after faulty gene prevents immune system working
- Allele inserted into retrovirus
- Initially caused leukaemia as inserted in the wrong place.
Gene Therapy- Liver disease
- Unable to breakdown ammonia so it builds up in the blood
- Low protein diet or transplant can help
- Someone died in trials so they were discontinued
Ethics of Genetic Manipulation- Microbes
Benefits- Insulin production -Produce vaccines -Antibiotics Risks- Antibiotic resistance -Used in warfare -Accidentally create worse pathogen
Ethics of Genetic Manipulation- Plants
Benefits- Pest and herbicide resistant -Golden rice -Drought resistant Risks- Resistant weeds -Enters food chain -Reduced biodiversity
Ethics of Genetic Manipulation- Animals
Benefits- Increased yield
-Spider goats
-Xeno transplants
Risks- Issue of welfare and ethics
Ethics of Genetic Manipulation- Humans
Benefits- SCID -Cystic Fibrosis Risks- Death from immune system -Long term effects -Treatment is continuous which is costly -Germline is illegal
