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SBI4UZ IB HL Biology Chapter 3.5 Genetic Modification (Gel electrophoresis…
SBI4UZ IB HL Biology Chapter 3.5 Genetic Modification
Polymerase chain reaction: artificial method of replicating DNA
1) Denaturation: heated to separate into two strands at 95C
2) Annealing: DNA primers attach to 3' ends, cooled to 55C
3) Elongation: DNA (taq) polymerase binds to primer, copying strand at 72C
Gel electrophoresis: and isolating proteins/DNA fragments based on size
Process
Samples placed in block of gel, electric current applied causing samples to move through gel (DNA is negative); smaller samples less impeded by gel and move faster
DNA Separation
Cut into fragments using restriction endonuclease; different samples generate different lengths
Fragments separate b/c DNA negatively charged due to phosphate group
Samples placed into agarose gel, fragment size calculated by comparing against known industry standards
Sequences identified by incorporating complementary radiolabelled probe and transferring sequences to membrane to be visualized via autoradiography
Protein Separation
Proteins folded into variety of shapes and have no clear charge
First treated w/ anionic detergent to linearize and impart uniform negative charge
Protein samples placed into polyacrylamide gel, sizes compared against industry standards
Separated proteins transferred to membranes, target proteins identified by staining
DNA profiling: technique by which individuals can be identified/compared via number of repeats of DNA elements at given satellite DNA locus
Process: DNA sample collected, amplified via PCR, satellite DNA cut w/ restriction enzymes, fragment length differs b/t individuals, fragments separated using gel electrophoresis
Forensic investigations: suspects should be 100% match w/ DNA sample taken from crime scene
Gene transfer: genes determine particular trait by encoding for specific polypeptide
Step 1 (isolating gene and vector): DNA isolated via centrifugation, gene of interest amplified via PCR, complementary DNA (lacking introns) generated from mRNA using reverse transcriptase, vector (DNA molecule acts as vehicle to carry gene of interest into foreign cell) commonly bacterial plasmids b/c of autonomous self-replication and expression
Step 2 (Digestion w/ Restriction Enzymes): gene and vector cut at specific recognition sites w/ restriction enzymes which cleave sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs) **sticky ends preferred w/ restriction endonucleases to ensure current insertion orientation, prevent vector reannealing
Step 3 (ligation of vector and insert): gene inserted into vector b/c sticky ends overlap via complementary base pairing, spliced together by DNA ligase to form recombinant construct (fusing sugar-phosphate backbones w/ phosphodiester bond)
Step 4 (selection and expression): recombinant construct introduced into host organism
GMO Debate
Pros
Improved nutritional values via proteins, vitamins, vaccines
Crops produced w/out allergens
Crops grow in arid conditions for better yield
Produce herbicides to kill pests
Improve food supply in poor countries
Longer shelf life
Less economic costs and carbon footprint
Cons
Adverse health reaction due to new proteins
Unknown effects of removal of traits
Limited biodiversity (increased competition w/ native species)
Super weeds from cross pollination
Patents restrict farmers from accessing GM seeds
Food with GM components not labeled
Diff. governments have different regulatory standards
Clones: groups of genetically identical organisms derived from single parent cell, requiring production of stem cells
Somatic Cell Nuclear transfer: method where cloned embryos produced using differentiated adult cells
1) somatic cells removed from adult donor, cultured
2) unfertilised egg removed and haploid nucleus removed to produce nucleated egg cell
3) enucleated egg cell fused w/ nucleus from adult donor to make diploid egg cell
4) electric current used to stimulate egg to divide, develop embryo
5) embryo implanted into surrogate uterus, develop into clone of adult donor
Natural cloning
Animal methods
Binary fission: parent organism divides equally in two
Budding: cells split off parent, generating smaller daughter organism which eventually separates
Fragmentation: new organisms grow from separated fragment
Parthenogenesis: embryos formed from unfertilised ova via production of diploid egg cells
Plant methods
Vegetative propagation where small pieces induced to grow independently b/c adult plants possess totipotent cells
Spore production
Stem cuttings: separated portion of plant stem that can regrow into new independent clone
Typically placed w/ lower nodes covered, upper nodes exposed; factors influencing successful rooting include:
Cutting position
Length of cutting (how many nodes remain)
Growth medium
Use/concentration of growth hormones
Temperature
Availability of water
Human methods
Monozygotic twins: zygote split into two identical cells each forming embryo (share 100% of DNA)
Dizygotic twins: unfertilised egg splits into two cells, each fertilised by different sperm (share 50% of DNA)
Artificial cloning: for purposes of reproduction or therapy (new cloned organism vs. organs/tissues for transplantation
Pluripotency in embryonic cells
Embryonic cells separated in lab, each group will form cloned organisms; can occur naturally to form monozygotic twins
Separation should happen ideally at 8-cell stage, which are then implanted into uterus to develop genetically identical clones
Somatic cell nuclear transfer (replacing haploid nucleus of unfertilised egg w/ diploid nucleus from adult donor)
Advantageous b/c the clone's traits will be known