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GENETIC MODIFICATION + BIOTECHNOLOGY - Coggle Diagram
GENETIC MODIFICATION + BIOTECHNOLOGY
CLONING/CLONES
Clones are groups of genetically identical organisms or a group of cells derived from a single original parent cell
Organisms that reproduce asexually will produce genetically identical clones
Additionally, mechanisms exist whereby sexually reproducing organisms can produce clones (e.g. identical twins)
Cloning multicellular organisms requires the production of stem cells (differentiated cells cannot form other cell types)
Stem cells can be artificially generated from adult tissue using a process called somatic cell nuclear transfer (SCNT)
Somatic cell nuclear transfer is a method by which cloned embryos can be produced using differentiated adult cells
Somatic cells are removed from the adult donor and cultured (these cells are diploid and contain the entire genome)
An unfertilised egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
The enucleated egg cell is fused with the nucleus from the adult donor to make a diploid egg cell (with the donor’s DNA)
An electric current is then delivered to stimulate the egg to divide and develop into an embryo
The embryo is then implanted into the uterus of a surrogate and will develop into a genetic clone of the adult donor
MONARC BUTTERFLY
In 1999, a preliminary study was conducted investigating the association between exposure to Bt corn pollen and survivor rates among monarch caterpillars:
Monarch caterpillars were fed milkweed leaves that had been dusted with pollen from Bt corn (to simulate spread via wind)
Growth and mortality rates were compared against caterpillars fed on non-dusted leaves or leaves dusted with non-GM pollen
Caterpillars exposed to Bt pollen were found to have eaten less, grew more slowly and exhibited higher mortality rates
Some scientists suggested that these results may lack validity as they do not accurately reflect natural conditions:
There were higher amounts of Bt pollen on the leaves than would be found naturally (e.g. rain would diminish build up)
Larva were restricted in their diet (in the field, larva could feasibly avoid eating pollen dusted leaves)
A second study was conducted comparing the survivor rates of monarch butterflies based on proximity to Bt corn fields:
There was no significant increase in mortality when monarch larva were placed in or near an actual Bt corn field
From this it was concluded that exposure to Bt pollen poses no significant risk to monarch butterfly populations
RISKS + BENEFITS OF GMO's
PCR
PCR can be used to amplify small amounts of DNA.
The polymerase chain reaction (PCR) is an artificial method of replicating DNA under laboratory conditions
The PCR technique is used to amplify large quantities of a specific sequence of DNA from an initial minute sample..
Each reaction cycle doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230).
Stages of PCR
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps. Once large quantities of DNA have been created, other laboratory techniques are used to isolate and manipulate the sequences
Denaturation – DNA sample is heated to separate it into two single strands (~95ºC for 1 min)
Annealing – DNA primers attach to the 3’ ends of the target sequence (~55ºC for 1 min)
Elongation – A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand (~72ºC for 2 min)
GEL EKECTROPHORESIS
Gel electrophoresis is a laboratory technique used to separate and isolate proteins or DNA fragments based on mass / size
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
Smaller samples are less impeded by the gel matrix and hence will move faster through the gel
This causes samples of different sizes to separate as they travel at different speeds
DNA Separation
Specific sequences can be identified by incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography (Southern blotting)
DNA samples are placed into an agarose gel and fragment size calculated by comparing against known industry standards
Fragments separate because DNA is negatively charged due to the presence of a phosphate group (PO43–) on each nucleotide
DNA may be cut into fragments using restriction endonuclease – different DNA samples will generate different fragment lengths
Protein Separation
Proteins may be folded into a variety of shapes (affecting size) and have positive and negative regions (no clear charge)
Proteins must first be treated with an anionic detergent (SDS) in order to linearise and impart a uniform negative charge
Protein samples are placed into a polyacrylamide gel and sizes compared against known industry standards
Separated proteins are transferred to a membrane and then target proteins are identified by staining with specific monoclonal antibodies (Western blotting)
DNA PROFILING
DNA profiling is a technique by which individuals can be identified and compared via their respective DNA profiles.
Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements called short tandem repeats (STRs)
As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA
DNA profiling is commonly used in criminal investigations (forensics) and to settle paternity disputes
Forensic Investigations:
Suspects should be a complete match with the DNA sample taken from the crime scene if a conviction is to occur
The number of loci used to generate a unique profile depends on the size of the population being compared
A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments
Fragment length will differ between individuals due to the variable length of their short tandem repeats
The fragments are separated using gel electrophoresis and the resulting profiles are compared
Paternity Testing: Children inherit half their chromosomes from each parent and thus should possess a combination of parental fragments In other words, all fragments produced in the child should also be produced by either the mother or father
GENE MODIFICATION THROUGH GENE TRANSFER
A gene determines a particular trait by encoding for a specific polypeptide in a given organism
Because the genetic code is (almost) universal, an organism can potentially express a new trait if the appropriate gene is introduced into its genome
The transfer of genes between species is called gene modification, and the new organism created is called a transgenic
Step 2: Digestion with Restriction Enzymes
In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites
Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from re-annealing without the desired insert
Step 3: Ligation of Vector and Insert
The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond
Step 1: Isolating gene and vector
DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression
These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters)
Other types of vectors include modified viruses and artificial chromosomes
Step 4: Selection and Expression
The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism
This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)
Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct
The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic
Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest
