gene tech and mutations Flashcards

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1
Q

DNA ligase

A

‘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.

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2
Q

use of vectors

A

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.

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3
Q

plasmid

A

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.

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4
Q

recombinant DNA tech steps

A

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.

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5
Q

Uses of recombinant DNA technology

A

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

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6
Q

Vaccines and recombinant DNA tech

A

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.

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7
Q

HepB virus process (recombinant DNA tech)

A

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.

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8
Q

Diabetes

A

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.

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9
Q

Type 1 diabetes

A

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.

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10
Q

Type 2 diabetes

A

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.

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11
Q

hyperthyroidism (overactive)

A

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.

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12
Q

Hypothyroidism (inactive pituitary)

A

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.

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13
Q

Hypothyroidism (iodine deficiency)

A

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.

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14
Q

Gene therapy

A

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.

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15
Q

gene therapy steps

A

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.

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16
Q

Type 1 diabetes gene therapy

A

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

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17
Q

Cystic fibrosis

A

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.

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18
Q

Cystic Fibrosis gene therapy

A

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.

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19
Q

Huntington’s disease

A

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.

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20
Q

huntingtons gene therapy

A

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.

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21
Q

Germline cell therapy

A

repair the gene in a fertilised egg, so that the required gene would be copied into each daughter cell at mitosis.
Results in elimination of the mutant gene from (1) person receiving treatment and from (2) their offspring - future generations. Genetic change can be inherited.

22
Q

Somatic cell therapy

A

Repair gene in normal body cell of patient.
results in elimination of mutant gene in individual only
Disorder still able to be passed onto offspring.

23
Q

Cell therapy ethical issues

A

playing god: manipulating factors such as gender, shortness, gender, intelligence (IQ), eye/hair colour - creating designer babies.
Embryos genes scanned + replaced

24
Q

cell replacement therapy

A

Stems cells are undifferentiated cells that are capable of repeated mitotic divisions for ling periods of time and given the right conditions, can differentiate into specialised cells.
any disorder involving loss of, or injury to, normal cells in potentially a candidate for CRT.
The replacement of damaged cells with healthy ones injected into patient.
Use embryonic stem cells (patients own stem cells)

25
Q

Parkinsons disease (PD)

A

Cause: genetic, affects dopamine producing neurons in the brain (basal ganglia of the midbrain)
Symptoms: shaking, slow movement, muscle stiffness, stooped posture and impaired speech. Affects older people, varying symptoms.
treatment: medication such as dopamine to increase levels - relieve symptoms and manage condition.

26
Q

Alzheimer’s diseases (AD)

A

Cause: brain cells die, abnormal plaques/tangles - toxic deposits. Neurons stop functioning from dementia. Age: 65+
symptoms: loss of memory, confusion, mood swings, aggression, withdrawal.
Treatment: drugs to slow degeneration by increase the level of NT - acetylcholine in the brain.

27
Q

Cell replacement therapy Parkinsons disease (PD)

A

aim: to replace dying neural tissue wt healthy tissue -> cure disease.
Steps:
1. Selection of source of cells. Induced pluripotent stem cells (from patient) -> differentiate under correct conditions into neural cells.
2. Transplantation into patient (method of introduction)
3. Place of introduction - brain
4. Dopamine levels replaced, providing symptomatic relief.

28
Q

Tissue engineering

A

aim: to restore healthy tissues or organs to avoid need for organ transplants or artificial implants
Steps:
1. Stem cell isolation (biopsy): use stem cells, taken from patient who needs tissues repair.
2. Cultured cells in Petri dish - induce them to grow (proliferation)
3. Seeding: introduce scaffold of natural or synthetic material -> cells cover scaffold. Add mechanical (electrical) and chemical stimulus - to stimulate cell growth - 3D tissue results. (Scaffold biodegradable)
4. Implantation of ‘engineered’ tissue into patient’s body at site that needs new tissue.
Developing: bone, skin, cartilage, adipose tissue.

29
Q

mutation

A

A change in a gene or chromosome leading to new characteristics in an organism.
2 types:
1. Gene mutation - changes in a single gene so that the traits normally produced by that gene are changed r destroyed.
2. Chromosomal mutation - all or part of a chromosome is affected.

30
Q

Mutant

A

an organism with a characteristic resulting from a mutation

31
Q

Cause of mutation

A

induced mutation - mutation caused by mutagen
Spontaneous mutation - occurs due to a random error in a biological process such as mitosis or meiosis.

32
Q

heritability of a mutation

A

Somatic mutation - a change occurring in a body cell. Since gametes aren’t affected, offspring is not affected. Involved in most cancerous growths.
Germinal (gremline) mutation - if reproductive cells are affected, the mutation can occur on the gametes and may then be passed on to the next, and subsequent generations. Individual who has the gametes is unaffected. If conception occurs involved mutated gametes, embryo usually aborted naturally. Eg. PKU, phenylketonuria, can arise from a mutation during the formation of gametes and can be passed on to offspring.

33
Q

effect of the mutation

A

missense
nonsense
Neutral
silent

34
Q

Missense mutations

A

cause a change in the amino acid, and therefore in the protein produced

35
Q

Nonsense mutation

A

change the base sequence to the code to STOP.
This means that the synthesis of the protein will stop, and so a shorter protein is produced that is unlikely to be able to fulfil its function.

36
Q

neutral mutations

A

Cause a change in an amino acid; however, the amino acid is of the same type and does not change the structure of the protein enough to change its function.

37
Q

silent mutations

A

Do not cause any change in amino acid, therefore in the protein produced.
possible as most amino acids are coded by more than one base sequence.

38
Q

Change in the DNA

A

point mutations
Frameshift
Mutations affecting larger sections: Duplication, Deletion, Inversion, Translocation and non-disjunction

39
Q

Point mutations

A

due to changes in a single nucleotide, one base is changed.
due to nucleotide being: inserted - a new nucleotide is added to the DNA strand, substituted - an existing nucleotide is replaced wt another one, wt a different base. Deleted - a nucleotide is removed from the DNA strand.

40
Q

Frameshift mutation

A

occurs when bases have been added or removed.
Affect the outcome of all DNA from that point on.
Not a frameshift if 3 bases added or deleted as DNA will code for one more or one less amino acid.

41
Q

Duplication (or insertion)

A

a section of chromosome occurs twice.

42
Q

Deletion

A

a piece of DNA is removed

43
Q

Inversion

A

Breaks occur in a chromosome and the broken piece joins back in, but the wrong way around.

44
Q

translocation

A

Part of a chromosome breaks off and is rejoined to the wrong chromosome

45
Q

non-disjunction

A

During meiosis, a chromosome pair does not seperate and so one daughter cell has extra and one has less.
referred to as aneuploidy, change in the chromosome number.

46
Q

Conditions due to mutations

A

gene mutations: Duchene muscular dystrophy, cystic fibrosis
Chromosomal mutations: trisomy (Down syndrome, trisomy 21)(Patau syndrom, trisomy 13)(Klinefelter syndrome, X or Y chromosome), monosomy (Cri-du-chat syndrome, partial monosomy 5)(Turner syndrome, missing X or Y chromosome)
lethal recessives: tay-sacks syndrome

47
Q

Duchene muscular dystrophy

A

arises from mutation in mom which is inherited by son, can also be from male zygote.
Disease results in a wasting of the leg muscles and later, the arms, shoulders and chest.
usually becomes apparent at age 3-5 when muscle weakness is evident.
Eventual death occurs due to failure of respiratory muscles.
Boys unlikely to live more than 20-25 years.

48
Q

Trisomy

A

result of non-disjunction, failure of one or more chromatids to seperate in second division of meiosis
Extra chromosome
partial trisomy - part of an extra copy is attached to one of the other chromosomes

49
Q

Monosomy

A

missing a chromosome
partial monosomy - if only part of the chromosome is missing

50
Q

Lethal recessives

A

cause of death of the embryo or foetus by a miscarriage or spontaneous abortion, or the early death of child.