gene tech and mutations Flashcards
DNA ligase
‘DNA-joining enzyme’ found in E.coli
ligation - the process of joining short strands of DNA during cell replication/division.
complementary bases must 1st join by forming hydrogen bonds.
Then DNA ligase joins the backbone: phosphate group at end of 1 strand to the sugar molecule at the end of another strand.
use of vectors
A vector is a DNA molecule that is used to carry DNA into a cell.
commonly used in recombinant DNA tech: 1. Bacterial plasmids, 2. Bacteriophage viruses (viral vectors)
Cloning - of vector (plasmid) -> many copies
plasmid returned to bacterium (host cells_
Treat bacteria -> multiply -> protein.
all cultured in vitro (culture dish, Ttube)
Other vectors - yeast, mammalian cells.
plasmid
Circular, double stranded units of DNA.
capable of replicating in a cell independent of chromosome DNA.
Gene of interest is inserted into plasmid.
Plasmid then returned to bacteria = recombinant bacterium.
recombinant DNA tech steps
1. Isolate the gene and it out using restriction enzyme.
2. Isolate a plasmid from a bacterial cell and cut it with the same type of restriction enzyme used in step 1.
3. Splice the human DNA into the plasmid using DNA ligase enzyme to join the sticky ends.
4. Treat the bacterium so it takes up the recombinant plasmid. Once this is successful, the bacterium will multiply so that either the human gene or the product of the gene can be used.
Uses of recombinant DNA technology
diagnosis and treatment of diseases and genetic disorders.
Manufacture of large quantities of pure protein:
1. Insulin - diabetes
2. Human growth hormone (HGH)-(children)
3. Follicle stimulating hormone (FSH) - infertility
4. Factor VIII blood clotting factor - haemophilia
Vaccines and recombinant DNA tech
1st vaccine made was hepatitis B vaccine 1986.
Inserted gene from hep virus into cowpox virus.
Hep B virus -> liver damage. Babys give 1st shot at birth.
Most vaccines today uses recombinant bacterium Ecoli.
HepB virus process (recombinant DNA tech)
1. Gene for surface antigen isolated (RE to cut)
2. Plasmid DNA cut with same restriction enzyme as virus DNA.
3. Splice viral DNA into plasmid (DNA ligase)
4. Introduction of recombinant DNA into yeast cell (vector)
5. Fermentation tank: when recombinant yeast cell divides -> new cells contain plasmid with gene to make Hep B antigen.
6. Gene allows yeast cells to produce the Hep B antigen protein.
7. Extraction and purification of Hep B antigen
8. Vaccine available for use.
Diabetes
is a disease characterised by abnormally high levels of blood glucose.
Body unable to maintain the Bg level in homeostatic balance.
a diabetic usually has hyperglycaemia - high bg.
Symptoms of hyperglycaemia: polyuria (inc. urine), polydipsia (inc. thirst), hunger, weight loss, tiredness.
Benefits of treatment: decreased risk of long term effects like: kidney failure, stroke, heart attack, nerve damage, amputations, blindness.
Type 1 diabetes
other names: diabetes mellitus, insulin dependant, juvenile onset diabetes.
Description: the pancreas is unable to produce insulin
cause: an autoimmune response= person’s immune system attacks/ destroys own beta cells of the islets of langerhans in pancreas. (Insulin not made). Body then burns fat as an energy source and Ketoacidosis occurs. Genetic factor may be involved.
age of onset: childhood
Treatment: insulin injections (x2 or x4 daily) cannot be taken by mouth because insulin is a protein and so would be digested, injected using syringe, programmable pump or insulin pump.
Type 2 diabetes
Other names: adult onset diabetes, non-insulin dependent.
description: cells/receptors resist effects of insulin. Pancreas does not produce enough insulin, or both.
Causes: body cells cannot respond to insulin. Lifestyle disease - people not physically active, overweight, obese. Risk increased: if overweight or obese, high Bp, high cholesterol, smoking, unhealthy diet - high in fat, sugar and low in fibre, decrease in physical activity.
age of onset: usually arises in people over 45 years (late onset diabetes)
Treatments: no cure, increased exercises, manage diet, monitor bgl, monitor Bp, often insulin therapy.
hyperthyroidism (overactive)
Description: over production of thyroxine (T4, T3) by thyroid gland (overactive). Most common type is grave’s disease. Increased metabolic rate, affect women more than men, peak 30-40yrs ratio 8:1 f:m.
causes: enlargement of thyroid gland. Caused by autoimmune disorder/response. Genetic disposition (not inherited). Cancer - causing overproduction.
Symptoms: (overstimulation of cells due to thyroxine levels). Leads to rapid heartbeat, weight loss, increase appetite, fatigue, inc. sweating, anxiety, feel too hot - heat intolerance, protruding eyeball in Grave’s disease also called exophthalmia due to inflammation.
treatments: 1. Anti-thyroid drugs to reduce thyroid function - block glands ability to use iodine. 2. Radio iodine therapy - drink radioactive iodine (thyroid cells take it up and die). 3. Surgery to remove all or part of gland.
Hypothyroidism (inactive pituitary)
Description: under production of thyroxine (underactive), results in low metabolic rate. Common in women age 65 above.
causes: problems wt thyroid gland, pituitary gland or hypothalamus. Most common cause: autoimmune attack on the thyroid gland = Hashimoto’s disease. Lack of iodine in diet. Surgery of thyroid gland due to cancer.
Symptoms: (under stimulation) leads to - weight gain, slow heart rate, fatigue, lack of energy, drowsy. Cold intolerance, swelling of the face and Goitre.
treatments: synthetic thyroxine tablets - levothyroxine - synthesised from chemicals in lab. Taken for rest of life. Once extracted from pigs.
Hypothyroidism (iodine deficiency)
description: under production of thyroxine.
Causes: lack of iodine in the diet preventing the thyroid gland from making enough thyroxine. Thyroid gland becomes enlarged in an effort to increase the hormone (pit. Produces more TSH - grows in size). Results in Goitre.
symptoms: slow HR, unexplained weight gain, fatigue, lack of energy. Cold intolerance and goitre.
Treatment: addition of extra iodine in diet. i) iodised table salt, ii) addition of iodine in bread.
Gene therapy
Aim: to treat or cure genetic (inheritable) diseases at their source. Focuses is on single gene disorders.
areas of possibility: 1. Replacing a mutated gene wt a new healthy gene. 2. Fixing or inactivating mutated genes. 3. Inserting a new gene that will fight the disease. 4. Making the immune system recognise diseased cells (eg. Cancer cells) and destroy them.
Concept: a vector can be used to deliver desired DNA/gene into a cell -> will be incorporated into cell nucleus and undergo transcription (mRNA) and translation (ribosomes) to produce a desired protein.
gene therapy steps
1. Isolate new healthy gene
2. Insert into viral vector -> into body (remove disease causing genes from virus)
1. Vector binds to cell membrane
2. Vector packaged into vesicle
3. Vesicle breaks down releasing vector
4. Vector binds to nuclear membrane
5. New gene enters nucleus.
Type 1 diabetes gene therapy
1. Gene for insulin inserted into a vector -> into pancreas
2. Vector used to ‘infect’ desired cells - alpha cells of islets of langerhans in pancreas.
3. These cells incorporate new DNA into their nucleus + undergo protein synthesis (transcription and translation) to produce insulin.
4. Body can function normally
Cystic fibrosis
cause: autosomal recessive genetic disorder (cc) 1:25 people carry gene.
Mutation to cystic fibrosis transmembrane regulator gene (CFTR). Single gene disorder.
symptoms: affects lungs, pancreas, liver and reproductive organs. Thick sticky mucous - clogs air passages, traps bacteria -> infections -> lung damage. Blockages in pancreas - enzymes required for digestion affected -> nutrition problems. Chloride transport is affected -> nutrition problems. Chloride transport is affected.
Diagnosis: at birth (heel prick test). If positive result, special low fat, high carbohydrate, high protein diet is advised, DNA sequencing.
treatment: digestive enzymes, antibiotics, inhaled medicines - airway clearance.
Cystic Fibrosis gene therapy
1. Gene for healthy (CFTR) inserted into a virus vector (adenovirus or liposome fat globule) -> into lungs
2. Vector used to ‘infect’ desired cells - lung cells
3. These cells incorporate new DNA into their nucleus + undergo protein synthesis (transcription and translation) to produce correct protein - to restore salt transport in cells + halt lung disease.
4. Lungs/Body can function normally.
Gene therapy logical Choice: 1. Single gene disorder. 2. Lung most severely affected organ - easy to access. 3. Disease slow to progress - lungs of newborn virtually normal. 4. Gene therapy can begin before lung damage occurs.
Huntington’s disease
Cause: genetic disorder autosomal dominant 50:50 chance of inheritance if parent carries.
mutation to the IT15 gene. Chr4 single gene disorder. 1:10000 people carry gene. Mutation to protein called Huntingtin - toxic to nerve cells in basal ganglia of brain. Neurodegenerative disorder. Symptoms appear middle age - 40yrs, it is a slow progressive disease. Midlife onset 30-50yrs of age.
Symptoms: physical, cognitive, emotional. Nerve cells in brain are damaged, involuntary flailing movements of muscles of arms and legs, uncontrolled jerking and twisting movements. Progressive dementia, loss of ability to think clearly, depression, mood swings, behavioural problems.
Diagnosis: family history, people at risk - genetic test available (on embryo) DNA sequencing. Then genetic counselling.
Treatment: drugs, physiotherapy. No cure. Reduced life expectancy 10-20yrs.
huntingtons gene therapy
Research on mice (brains) shown it can be possibly be effective on humans.
a modified virus being tested to deliver a corrective gene into brain cells that boosts a natural shield against the effects of the defective Huntingtin protein - research on rats and primates -> clinical trials on humans now.
