4.5 application of reproduction and genetics Flashcards
1
Q
what were the main goals of the human genome project?
A
- identify all the genes in the human genome and identify their loci
- determine the sequence of the 3.6billion bases present in the human genome and store in databases
- consider the ethical, social and legal issues that arise from storing this informatiom
2
Q
what did the human genome project find?
A
- the number of genes present in the human genome is around 20,500
- there are large numbers of repeating sequences called STRs (short tandem repeats)
3
Q
- the human genome project began in 1990
- it took 10 years to complete
- but the analysis of all the sequences obtained took much longer
A
4
Q
what method of sequencing did the human genome project use?
A
- sanger sequencing
5
Q
how does sanger sequencing work?
A
- by sequencing small fragments of DNA around 800 bases in length created by the use of restriction enzymes
- DNA polymerase was then used to synthesis complementary strands using the polymerase chain reaction
- 4 reactions were carried (one for A, T, C and G), each containing complementary nucleotides marked with a radioactive marker, but a proportion of the nucleotides used in each reaction had been altered (stop nucleotides)
- when they were incorporated into the complementary strand, further synthesis was prevented
- when the result for all the reactions for each nucleotide are run out side by side on agarose gel using electrophoresis, and the resulting gel exposed to x-ray film to detect the radioactive signal, the sequence can be determined by reading the banding pattern because electrophoresis separates DNA fragments according to size
6
Q
what are restiction enzymes?
A
bacterial enzymes that cut DNA at specific base sequences
7
Q
what is polymerase chain reaction (PCR)?
A
a technique that produces a large number of copies of specific fragments of DNA, rapidly
(used to rapidly amplify fragments of DNA)
8
Q
what is gel electrophoresis?
A
- a method of separating DNA fragments according to size using an electric current
9
Q
- the sanger method is very slow, taking days to accurately sequence a few thousand bases
- with the introduction of Next Generation Sequencing (NGS), entire genomes can be sequenced in hours
A
10
Q
what did the 100K genome project aim to do?
A
- sequence 100,00 genomes from healthy individuals and patients with medical conditions across the uk to establish any variance in their base sequence and identify if there is genetic correlation
- enables scientists to study variation in the human genome
- improve the accuracy of diagnoses
- better predict the action of drugs
- improve the design of drugs
- find new ways of treating genetic diseases
- explore the possibility of tailoring therapies to treat a disease in an individual lerson
11
Q
what did the 100k genome project use?
A
- NGS (next generation sequencing)
12
Q
when was the 100k genome project launched?
A
in 2012
13
Q
what are some ethical concerns with the human genome and 100k projects?
A
- if a patient had a genetic predisposition to a particular disease, should this information be passed to life or health insurance companies?
- if ancestral relationships are determined, this could be used to socially discriminate against people
- if genetic diseases are identified, this has an implication for the parents and children of those diagnosed. if children are screened, when should they be told if they have a predisposition for a disease
- could screening embryos be extended from genetic diseases to desirable traits
- how to ensure safe storage of patient data
14
Q
what has allowed us to determine evolutionary relationships e.g how closely related we are to primates?
A
other organisms having had their genomes sequenced
15
Q
- the mosquito Anopheles gambiae which is responsible for transmitting malaria to around 200 million people annually, has also had its genome sequenced in attempts to tackle insecticide resistance in the vector
A
16
Q
gene-editing in combating malaria:
A
- in 2015 gene-editing technology was used to produce a genetically modified mosquito that could produce antibodies to the Plasmodium parasite that it transmits
- whilst the mosquito wont be released into the wild, it is a step forwards in the control of malaria
(other attempts to control malaria have focused on the parasite, Plasmodium
- it too has developed resistance to many of the drugs used to treat it, but it’s hoped that the sequencing of its genome will allow for the development of new drugs
17
Q
what can be made rapidly using the polymerase chain reaction (PCR) technique?
A
- large number of copies of specific fragments of DNA
18
Q
from each strand of DNA, it’s possible to produce how many copies in a few hours?
A
over a billion copies in a few hours
(30 cycles of PCR will produce 2^30 copies of DNA - which is just over 1 billion)
19
Q
what does PCR require?
A
- a heat stable DNA polymerase isolated from the bacterium Thermus aquaticus, which lives in hot springs
- short single-stranded pieces of DNA called primers (6-25 bases long)
- deoxyribonucleotides containing the four different bases
- a buffer
20
Q
what are primers? and what are they used for in PCR?
A
- are a short, single strand of DNA
- between 6-25 bases long
- they act as a start point for the DNA polymerase to attach
- are complementary to the start point on the DNA strand of interest
21
Q
during PCR, what is used to rapidly change the temperature?
A
a thermocycler
22
Q
what are the stages of PCR?
A
- heat to 95°C to separate the DNA strands by breaking the H bonds between the two complementary DNA strands
- cool to 50-60°C to allow the primers to attach by complementary base pairing (annealing)
- heat to 70°C to allow the DNA polymerase to join complementary nucleotides (extension)
- repeat 30-40 times
23
Q
what are some of the limitations of PCR?
A
- any contamination is quickly amplified
- DNA polymerase can sometimes incorporate the incorrect nucleotide (about once every 9000 nucleotides)
- only small fragments can be copied (up to a few thousand bases)
- the efficiency of the reaction decreases after about 20 cycles, as the concentration of reagents reduce, and products builds up
24
Q
what are short randem repeats (STR)?
A
- short sections of DNA found in the non-coding regions of the genome that show great variability in the number of times they repeat from individual to individual, so can be used to produce a genetic fingerprint
25
- a breakthrough came in the mid-1980s when it was found that they were able to use many variable regions of DNA which did not code for amino acids called short tandem repeats (STR) regions, to produce a genetic fingerprint
- what were these regions called?
- microsatellites
(and there are thousands of them scattered throughout the chromosomes)
- the number of times that these regions are repeated gives individuality
26
27
- PCR is then used to amplify specific microsatellite sequences from very small samples of DNA left at a crime scene
28
- an example of a STR is D7S280, a repeating sequence found on human chromosome 7
- the tetrameric repeat sequence of D7S280 is ‘gata’
- different alleles of this locus have from 6-15 tandem repeats of the ‘gata’ sequence, so the more times it repeats, the larger the fragment of DNA will be
- in an example, ‘gata’ is shown 13 times, so the size of the DNA fragment produced will be 13x4 bases = 52 bp
- currently 10 different microsatellite sequences are used to build up a unique fingerprint in uk
29
genetic fingerprinting:
- when the different-sized fragments are visualised by gel electrophoresis, a unique banding pattern is created
- to visualise DNA, what is often used?
- ethidium bromide
- as it intercalates with DNA (inserts between the base pairs) and fluoresces under ultraviolet light
30
what is the gel used in gel electrophoresis made from?
agarose (a polysaccharide which is the main constituent of agar) which contains pores in its matrix
31
how does gel electrophoresis separate DNA fragments according to size?
- DNA samples are placed at one end and a voltage is applied across the gel
- DNA is attracted to the positive electrode due to its negative charge on the phosphate groups
- smaller fragments migrate more easily through the pores in the gel and so travel further than large fragments in the same time
32
how can the size of fragments in gel electrophoresis be estimated?
- if a sample of known DNA sized fragments (called a DNA ladder) is run at the same time as the samples
33
what can be used to find DNA sequences of interest within DNA fragments?
DNA probes
34
35
what are probes?
- short piece of single stranded DNA that is labelled with a fluorescent or radioactive marker
- designed to be complementary to part of the sequence of interest
36
how can DNA probes be used to find DNA sequences of interest within DNA fragments?
- when the probe is washed over the gel, it binds to the exposed complementary nucleotides by a process known as DNA hybridation
- the DNA fragment which contains the sequence of interest is then identified by its fluorescence or radioactive signal
37
how do you detect a radioactive signal?
- the DNA from the gel is transferred to a nylon membrane, and the membrane is then exposed to x-ray film, producing an autoradiograph
38
what is DNA hybridisation?
- single-stranded DNA molecules anneal to complementary DNA
39
- DNA profiling is a non-invasive procedure requiring hair samples, or a mouth swab to collect enough DNA which can be further amplified by PCR
40
what are some used of DNA profiling (from genetic fingerprinting)?
- provide forensic evidence to identify or rule out suspects/to identify human remains
- paternity testing - here the genetic fingerprint of the child is composed of elements of the fingerprints of both parents. it has also been used to identify siblings
- screening for hereditary diseases
- selection for clinical trials
- phylogenic studies where relatedness of species can be investigated to suggest evolutionary links
41
what are some uncertainties/issues with genetic fingerprinting?
- DNA profiling cannot guarantee a match: at best a genetic fingerprint has a 1 in 1 billion chance that someone else could have the same profile, which still leaves some uncertainty
- ethical and legal concerns exist over the storage of DNA profiles by agencies such as the police or health insurance providers, and the safe storage of personal data
- DNA evidence in criminal cases is often relied upon too much to prove guilt, instead of supporting other evidence: a positive DNA sample from a crime scene may strongly indicate that a particular individual was present, not that they necessarily committed the crime
42
what does genetic engineering do?
- allows genes to be manipulated, altered or transferred from one organism or species to another, making a genetically modified organism (GMO)
(it is the modification of the genome of an organism by the insertion of a desired gene from another organism. this enables the formation of organisms with beneficial characteristics)
43
what are some examples of how genetic engineering has been used successfully?
- to produce insulin by inserting the human insulin gene into bacteria
- to produce disease resistant crops
- and with some success to reduce the symptoms of some genetic diseases e.g Duchenne muscular dystrophy (DMD)
44
when the genetic material from two species is combined, the result is _____
recombinant DNA
45
if donor DNA is inserted into another organism, the organism becomes ____
transgenic
46
what are the main tools used in genetic engineering?
restriction enzymes (restriction endonucleases)
47
what is recombinant DNA?
- DNA produced by combining DNA from two different species
48
what is a transgenic organism?
an organism that has been genetically modified by the addition of a gene or genes from another species
49
what are restriction enzymes named after?
the bacteria they have been isolated from
e.g EcoR1 comes from E.coli
50
what are restriction enzymes?
- bacterial enzymes that cut up any foreign DNA which enters a cell
51
when a restriction enzyme makes a cut, what are the two types of cuts it could be?
- STICKY CUTTERS - the cut may be staggered - and there are short, single-stranded fragments at either end - the overhangs are called ‘sticky ends’
- BLUNT CUTTERS - the cut may be blunt - the enzymes cut DNA between specific base sequences which the enzymes recognise and do not leave a ‘sticky end’ - these are called ‘blunt cutters’
52
before the introduction of PCR, how could DNA be amplified?
- by inserting it into a bacterial plasmid
- when the bacterium containing the plasmid divides, the plasmid (and its inserted DNA) is copied
53
- today, inserting genes into bacterial plasmids is used more to express the gene concerned and to collect the product made e.g human insulin
- genes of interest ate usually identified by the use of DNA probes, and cut out from the sample of DNA using restriction enzymes
- many eukaryotic genes contain introns (non-coding regions), so this method would also remove the introns and the gene would not be expressed
54
steps of using a restriction enzyme to insert a gene into a plasmid:
- the bacterial plasmid contains two mark genes: the first one is for ampicillin resistance so that any bacteria that contain the plasmid can grow on agar plate with ampicillin on it, and their growth is used to confirm that bacteria have taken up the plasmid
- the second marker uses a gene which is rendered non-functional if DNA is successfully inserted into it, and is used to confirm insertion of target gene
- the plasmid is cut with a restriction enzyme to open the plasmid
- the foreign DNA or gene is cut with the same restriction enzyme to ensure complementary sticky ends
- DNA is inserted using DNA ligase enzyme which joins the sugar-phosphate backbones of the two sections of DNA together
- to ensure that bacteria have a plasmid with the donor gene in, the the second marker gene is used, e.g Lac Z gene
- the Lac Z gene metabolises x-gal turning it from colourless to blue (and hence containing inserted DNA) will appear blue if x-gal is spread on the plate as they are unable to metabolise it
55
what is DNA ligase?
a bacterial enzyme that joins sugar-phosphate backbones of two molecules of DNA together
56
what is the function of the two marker genes in bacterial plasmids?
1. for ampicillin resistance so any bacteria that contain the plasmid can grow on an agar plate with ampicillin on it
2. uses a gene which is rendered non-functional if DNA is successfully inserted into it, and is used to confirm insertion of target gene
57
what are the two ways of isolating/locating a gene
- using restriction endonuclease
- reverse transcriptase
58
what are reverse transcriptases?
enzymes that produces complementary/copy DNA (cDNA) from an mRNA template
59
how are genes isolated using reverse transcriptase? (e.g the insulin gene)
- extract β cells from the pancreas (as has high proportion of mRNA that codes for insulin)
- reverse transcripted using reverse transcriptase
- makes DNA for insulin
- (human regulator DNA (controls gene expression) replaced with bacterial regulator DNA)
- DNA polymerase makes it double stranded
- purified
(as prokaryotes dont have introns in their DNA, this procedure gives introns-free DNA for insertion)
60
do prokaryotes have introns in their DNA?
no
61
what are some advantages of genetically engineered bacteria?
- allows production of complex proteins or peptide which cannot be made by other methods
- production of medicinal products e.g human insulin - far safer than using hormones extracted from other animals or donors
- can be used to enhance crop growth - GM crops
- GM bacteria have been used to treat tooth decay as they outcompete the bacteria which produce lactic acid that leads to dental caries
62
what are some disadvantages of genetically engineered bacteria?
- it is technically confusing and therefore very expensive on the industrial scale
- there are difficulties involved in identifying the genes of value in a huge genome
- synthesis of required protein may involve several genes each coding for a polypeptide
- treatment of human DNA with restriction endonuclease produces millions of fragments which are of no use
- not all eukaryote genes will express themselves in prokaryote cells
63
what are some hazards of genetically engineered bacteria?
- bacteria readily exchange genetic material e.g when antibiotic resistance genes are used in E.coli, these genes could be accidentally transferred to E.coli found in the human gut, or other pathogenic bacteria
- the possibility of transferring oncogenes by using human DNA fragments thus increasing cancer risks
64
making disease-resistant transformed plants:
- plasmid extracted from the bacteria
- restriction endonuclease is used to cut the plasmid and remove the tumour-forming gene
- a section of DNA containing a gene for disease resistance is located and isolated using the same restriction endonuclease
- the gene is inserted into the plasmid, replacing the tumour-forming gene. DNA ligase is used to join the donor and vector together
- the bacterial cell is introduced into the plant cell. the bacterial cell divides and gene is inserted into the plant chromosome
- transgenic plant cells are grown in tissue culture and transformed plants are regenerated
65
what are some benefits of GM crops?
- increased growth rates/greater crop yields
- improved nutritional value e.g high vitamin A precursor content in Golden Rice
- increased pest resistance / reduces need for pesticides
- ease of management e.g use of weed killers on resistant crops
- tolerance of unfavourable conditions e.g drought-resistant cotton and corn crops are being developed
- longer shelf-life of products
- reduces need for land clearing
66
what are some concerns of GM crops?
- genetic contamination / cross-pollination could result in herbicide-resistant weeds
- misuse of pesticides e.g overuse of the weed killers on resistant roundup
- control of agriculture, by corporations, e.g the control of seed supplies to farmers
- reduction in biodiversity
- unknown effect on health
- may increase costs for farmers
67
what are some hazards of GM crops?
- threats to biodiversity from the transfer of GM pollen to wild plants which can change natural gene pools. this may result in a reduction in biodiversity
- unknown effects of eating new protein produced in crops
68
what do genetic diseases involve?
- single cell conditions:
• Duchenne muscular dystrophy
• cystic fibrosis
- chromosomal disorders:
• Down’s syndrome
- multifactorial conditions:
• where faulty genes are part of the cause e.g Alzheimer’s disease
69
what does gene screening for genetic conditions allow for?
- accurate diagnosis and treatment
- identification of people at risk of preventable conditions
- pre-symptomatic testing for adult-onset disorders e.g alzheimer’s disease
- can help families to plan to avoid passing on conditions to children
70
what can gene screening involve testing?
- can involve testing:
• parents
• IVF embryos prior to implantation
• foetuses during pregnancy (pre-natal testing)
• newborns
71
- gene screening involves sessions with a genetic counsellor so that the implications are fully understood
72
what are some implications with gene screening?
- concerns exist over the storage and use of genetic test information
- e.g provision of health care or life insurance
- and that people may be discriminated against
73
what are some examples of GM crops?
- insect-resistant crops using the gene which codes for a toxin from the bacteria Bacillus thuringiensis (Bt)
- crops tolerant to herbicides
- crops with stacked traits of both Bt insect resistance and herbicide tolerance
- virus resistant crops
74
what is the most widely grown genetically modified crop?
soya
75
76
what is the aim of gene therapy?
- to treat a genetic disease by replacing defective alleles in a patient with copies of a new, functional DNA sequence
- but treatment can also involve replicating the function of genes using drugs
77
what are two methods of gene therapy?
- somatic cell therapy
- germ line therapy
78
what happens in somatic cell therapy?
- the therapeutic genes are transferred into the somatic (body) cells, of a patient
- any modifications and effects will be restricted to the individual patient only, and will not be passed on through gametes
- DNA is introduced into target cells by a vector e.g plasmid or virus
- e.g in the use of liposomes containing copies of the normal allele, to treat cystic fibrosis
79
what happens in germ line therapy?
no
- only germ line therapy is, but it is highly controversial
80
what is exon skipping?
- a type of treatment
- by introducing a ‘molecular patch’ over the exon with the mutation making the gene readable again
- a shorter form of the protein is formed, but one that is thought to be more functional than the untreated version
81
what’s an example of a condition treated by exon skipping?
- Duchenne muscular dystrophy (DMD)
82
is somatic cell therapy permanent?
no
- only germ line therapy is, but it is highly controversial
83
- cystic fibrosis:
- caused by a recessive allele that codes for a mutated form of the cystic fibrosis trans-membrane regulator (CFTR)
- the result is that the membrane protein is unable to transport chloride ions out of cells, and mucus found covering the epithelial tissues remains thick and sticky because the water potential is not lowered by the presence of chloride ions which draw water into the mucus by osmosis
- patients with cystic fibrosis therefore suffer from thickened mucus which blocks bronchioles and alveoli leading to recurrent infections
- mucus also blocks pancreatic ducts leading to poor digestion of food
84
what is a treatment of cystic fibrosis using gene therapy?
- involves insertion of the healthy CFTR gene into a liposome
- which is then administered by means of an aerosol
- the liposomes fuse with cell membranes lining the bronchioles allowing DNA to enter the cell and be transcribed
- as epithelial cells are continuously replaced, this is only a treatment and has to he repeated
85
what is a liposome?
a hollow phospholipid sphere used as a vesicle to carry molecules into a cell
(can contain the gene that is being introduced into a cell - acts as a vector)
86
what is the general effectiveness of gene therapy?
- results are variable
- and gene therapy often has to be repeated, so its not a cure
- the limited success is due in part to the plasmid often not being taken up, and when it is, the gene it contains is not always expressed
87
what is genomics?
- the study of the structure, function, evolution and mapping of genomes
- e.g the Human Genome and 100k projects
88
genomics should enable healthcare to be improved, by:
- more accurate diagnosis of diseSe
- better prediction of the effect of drugs and improved design of drugs. individual patients metabolise drugs in different ways, so it’s important to know whether a drug will be effective, and if so what the safe effective dose would be
- new and improved treatments for disease as a result of better understanding the biochemistry of diseases i.e the faulty proteins produced
(with the introduction of NGS technology it may be possible to look at tailoring therapies to individual patients where an individual could have a unique treatment for a common disease)
89
- the first licensed engineered tissue in 1998 was an artificial skin called ‘Apligraf’ used in place of skin grafts for burn patients
90
how is tissue engineering done? (e.g for making artificial skin in place of skin grafts for burn patients)
- fibroblasts were removed from skin cells and their life extended by elongating the telomeres present that usually shorten with successive cell division and therefore limit cell mortslity
- these cells were ‘seeded’ onto a scaffold, an artificial structure which can support growth of a 3D tissue
- scaffolds must allow diffusion of nutrients and waste products, allow cells to attach and move, and be able to be degraded and absorbed by the surrounding tissues as it grows
91
what is tissue culture to grow?
large numbers of genetically identical cells quickly from a single parent cell
92
what is tissue culture used to grow large numbers of genetically identical cells quickly from a single parent cell referred to as?
therapeutic cloning
93
what is the main advantage of therapeutic cloning?
- if patient’s cells are used, rejection of the tissue is unlikely
94
what conditions are needed for the tissue culture process?
- adequate oxygen and nutrients must be provided
- optimal conditions e.g temperature and humidity must be maintained
95
is reproductive cloning of humans involving whole organisms legal in the UK?
no
- it’s prevented by law in the uk
96
what are stem cells?
- undifferentiated/unspecialised cells which can differentiate into a range of different specialised cell types
97
what are the two main sources of stem cells?
- from embryos (embryonic stem cells)
- adult stem cells e.g bone marrow
98
are adult stem cells ‘true’ stem cells?
- no
- they give rise to new blood cells, but they are not ‘true’ stem cells as they are pluripotent and cannot differentiate into all types of cell like totipotent stem cells
99
what can stem cells be used for specifically?
- regenerate tissues and organs
- e.g pancreatic cells that fail to release sufficient insulin in patients with diabetes, damaged spinal cord cells or skin replacement for burn victims
- also be used to screen new drugs
- and develop model systems to study growth and birth defects
100
what are some advantages of stem cells?
- can be produced quickly
- produced on a large scale
- production of genetically identical cells for transplant, reducing risk of rejection
101
what are some disadvantages of stem cells?
- in mammals, the technique is very expensive and unreliable
- in plants, disease or entry of pathogens may cause problems
- inadvertent selection of disadvantageous alleles
- long-term / unforeseen effects such as premature aging
- ethical issues associated with obtaining stem cells from embryos and the cloning of humans tissues and organs
102
what is DNA sequencing?
identifying the base sequence of a DNA fragment
103
outline the potential benefits of the human genome project
- allows for the development of targeted, personalised medical treatments and greater accuracy of diagnosis
- increased opportunities for screening genetic conditions and early detection of disease
- enables the study of incidence of mutation in different genes
104
what is the 100k genome project?
- a uk government project that aims to study variation in the human genome amongst 100,000 uk citizens
- it uses Next Generation Sequencing (NGS)
105
describe Next Generation Sequencing (NGS)
- a faster, cheaper and more accessible method of sequencing that can sequence an entire genome in a few hours
106
describe genetic counselling:
- service that provides information and advice to people affected by or at risk of genetic diseases
- helps individuals and families to make informed decisions
107
what is genetic screening?
- testing individuals for certain faulty alleles
- used to detect disorders such as cystic fibrosis, Huntington’s disease and thalassemia
108
outline the potential disadvantages of genetic screening:
- screening for conditions such as cancer and Alzheimer’s disease only indicates increased risk - may cause unnecessary stress and anxiety
- there is no cure for many of the conditions - more stress
- what happens to the test result data? discrimination from employers or insurance companies? misuse of information
- risk of false positives or false negatives
- who should be screened? limited funds and time
- screening embryos could lead to ‘designer babies’
109
give some examples of organisms other than humans whose genomes have been sequenced
- chimpanzees and other primates
- Anopheles gambiae, the mosquito
- Plasmodium parasite
110
how has sequencing the genome of the mosquito, Anopheles gambiae, been useful to humans?
- Anopheles has developed insecticide resistance
- sequencing has enabled the development of chemicals that make Anopheles susceptible to insecticides
- (or perhaps that the resistance genes can be edited out)
111
outline the advantage of sequencing the genome of the plasmidium to humans
- plasmodium has developed multi-drug resistance
- enables the development of more effective drugs
112
what are exons?
coding regions of DNA
113
what are introns?
non-coding sequencing of DNA
114
what are STRs?
- short tandem repeats
- sections of repeated nucleotides within introns that produce variation in individuals
115
what techniques can be used to produce a genetic fingerprint?
- PCR
- gel electrophoresis
116
describe the reaction mixture in the first stage of PCR
- contains the DNA fragment to be amplified, primers that are complementary to the start of the fragment, free nucleotides to match up to exposed bases, and Taq DNA polymerase to create new DNA
117
what is Taq DNA polymerase?
- a thermally stable enzyme that synthesises a double-stranded molecule of DNA from a single template strand using complementary nucleotides
118
summarise the process of amplifying DNA fragments using PCR
- heated (95°C) to break hydrogen bonds between DNA strands
- cooled (50-60°C) to allow primers to bind - annealing
- heated (70°C) to activate Taq DNA polymerase and allow free nucleotides to join
- new DNA acts as a template for next cycle
119
summarise the process of using restriction enzymes to produce DNA fragments:
- gene identified using gene probe
- restriction endonucleases cut DNA at specific palindromic sequences producing sticky ends
120
summarise the process of using reverse transcriptase to produce DNA fragments:
- mRNA complementary to the target gene used as a template
- reverse transcriptase synthesises cDNA (complementary DNA)
- mixed with free nucleotides which match up to their base pairs. DNA polymerase joins nucleotides forming second strand
121
outline the advantages of using reverse transcriptase to produce cDNA
- dont have to locate the gene
- gene not cut into non-functional fragments by restriction enzymes
- introns not present in cDNA
- doesnt require post-transcriptional processing to produce functional mRNA
122
summarise the process of inserting a DNA fragment into a vector:
- plasmid (circular DNA from bacteria) used as the vector
- plasmid cut using the same restriction enzymes as the DNA, so that the sticky ends are complementary
- DNA ligase joins the fragment and plasmid together
123
describe how antibiotic-resistant genes are used in the identification of recombinant bacteria
- antibiotic resistance genes can be inserted into the plasmids at the same time as DNA fragment
- the transformed cells are then placed on a plate with antibiotics
- only the cells that successfully took up the vector will grow
124
give an application for genetic modification of bacterial cells
- human gene for insulin production can be inserted into a vector, so that the bacterial cell will produce insulin
- useful in medicine e.g. treatment of diabetes
125
outline the disadvantages of using recombinant DNA to make human products
- identifying the required gene may be difficult
- some eukaryotic genes can’t be expressed in prokaryotes
- antibiotic-resistance genes could be transferred to pathogenic bacteria
- expensive
126
what are GM organisms?
- organisms that have had their genome altered
127
differentiate between somatic cell therapy and germ line therapy
- somatic = allele introduced to target cells only, short-term, must be replaced
- gem line = allele introduced to embryonic cells so it is present in all resultant cells, permanent, passed onto offspring
128
what is a vector?
- a carrier used to transfer a gene from one organism to another e.g plasmid or virus
129
what is Duchenne muscular dystrophy (DMD)
- a recessive, sex-linked form of muscular dystrophy
- affecting up to 1 in 3500 live male births
- caused by a mutation in the dystrophin gene resulting in the failure to produce dystrophin (a structural component of muscle tissue)
- the result is severe wasting of the muscles and suffrtrs are often wheelchair bound by the time they reach their teenage years
- life expectancy is only 27
- characterised by muscle degeneration and weakness
130
what is the cause of DMD?
- it is caused by one or more mutations in the dystrophin gene that prevent the production of dystrophin
131
outline how DMD can be treated using gene therapy:
- healthy gene inserted into vector (e.g virus)
- vector inserted into muscle tissue
- virus delivers gene to muscle cells
- new gene incorporated into DNA of cell
- transcription and translation of gene produces normal dystrophin protein
- symptoms of DMD alleviated
132
what is drisapersen?
- an experimental drug that aims to treat DMD by exon skipping
133
explain how drisapersen works:
- it introduces a ‘molecular patch’ over the mutated exon, enabling the gene to be read
- a shorter, more functional type of dystrophin is synthesised
134
discuss the ethical issues surrounding the use of gene therapy:
- health implications - may produce an immune response, activation of oncogenes etc
- issues surrounding if it’s right to alter the genotype of an unborn child
- issues surrounding what conditions should be treated using gene therapy
- could lead to healthcare inequalities
- expensive - money could be better spent elsewhere
135
what is tissue engineering?
- an extension of gene therapy that aims to replace, repair or improve biological function by replacing organs and tissues
(the study of the growth of new connective tissues, or organs, from cells and a collagenous scaffold to produce a fully functional organ for implantation back into the donor host)
136
what are the ethical issues related to the use of stem cells from embryos?
- embryos used to provide stem cells are destroyed which is seen as unethical and a waste of potential human life
- could lead to the ‘farming’ of embryos for stem cells
- may lead to the reproductive cloning of humans
- the technology could be used for non-medical purposes
137
what did the Human Genome project aim to do?
- help us understand diseases to improve their diagnosis and treatment
- identify mutations linked to different forms of cancer
- improve design of medication
- more accurately predict the effects of drugs
- improve disease risk assessment
- advance forensic applied sciences
- use it in bioarcheology, anthropology and evolutioon
138
compare sanger sequencing and NGS
sanger sequencing:
- a large amount of template DNA is needed for each read - several strands of template DNA are needed for each base being sequenced
NGS:
- cheaper, quicker, needs significantly less DNA
- is more accurate and reliable
- a sequence can be obtained from a single strand
- automated, uses less resources and faster, so costs less
- includes built in repeats and because of the shorter time and lower costs more repeats can be made to increase reliability
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what are the ways in which NGS is quicker than Sanger sequencing?
- in NGS, the chemical reaction is combined with the sequencing
- in sanger sequencing, these are two separate processes
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what makes the 100k genome project possible?
- due to the reduced cost and time using NGS
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what are some differences between the human genome project and 100k project?
- the 100k project is only looking at genes involved with the development of disease
- the data isnt anonymous in the 100k project
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what are some advantages of genetic screening that can now be done following the results of the human genome project?
- can identify carriers of genetic diseases
- can provide genetic counselling
- can screen embryos prior to implantation
- assess future risk of developing a condition
- can check a foetus during pregnancy to detect the presence of disorders such as cystic fibrosis, Huntington’s disease and thalassaemia
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what is a benefit to sequencing the genome of chimpanzees and other primates?
- benefit in determining evolutionary relationships between primate groups
- may also benefit in aiding efforts to conserve these groups of animals
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malaria:
- malaria is caused by a protoctistan parasite, Plasmodium
- and is transmitted by a vector, the mosquito Anopheles gambiae
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what are some ethical issues with trying to gene edit mosquitos?
- it may alter the ecology of the area having a knock-on impact on other organisms that depend on mosquitoes for food
- it could be debated that this technique could lead to extinction of the mosquitoes by ,man which could be considered morally wrong
- the long-term effects of organisms genetically modified in this way on the ecology of an area are unknown
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- a gene is composed of regions that are translated X ones and influences which contain - exons and introns, which contain repeating base sequences between exons that are not translated
- it is the number of repeats of these base sequences in introns that is variable between individuals
- these blocks of repeated base sequences in introns are called short tandem repeats (STRs)
- in a genetic fingerprint a variety of these STRs are analysed to produce a unique pattern based on the number of times each STR was repeated
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what is a DNA ladder?
- markers of known length are run alongside the fingerprint DNA to give fragment size and position (in genetic fingerprints)
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- in genetic fingerprints, sometimes the lines can be quite close together or blurry making it difficult to make conclusions
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what are some issues with using restriction endonucleases to isolate a gene?
- the human genome is massive, and the enzyme will cut at a large number of places
- it is important to know how large the gene is in order to be able to isolate it with gel electrophoresis
- the base sequence on each side of the gene need to be known in order to use the correct type of restriction endonuclease and not to cut the gene in the middle somewhere
- usually the gene would be inserted into a plasmid and then transformed into a bacterium
- the bacteria will translate the gene and make the protein
- bacterial genomes do not have introns so they lack splicing methods, so unedited human genes may not be transcribed and translated
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what is an advantage of using reverse transcriptase to isolate a gene?
- mRNA is abundant in the cells producing the protein
- the mRNA has already undergone splicing, so introns have been removed
- there is no chance that the restriction enzyme can cut the gene and make non-functional fragments because they are not needed in this method
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genes can be cut out of human chromosomes using _______ enzymes
restriction endonuclease
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human genes have ____, but bacteria dont
introns
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cDNA can be made using mRNA as a template and the enzyme ______
reverse transcriptase
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cDNA can be made into double stranded DNA with _____ enzyme
DNA polymerase
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using reverse transcriptase means the gene produced lacks ____
introns
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specific base sequences are cut by a ______
restriction endonuclease
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what enzyme joins the two pieces of DNA together in plasmids when making recombinant DNA?
DNA ligase
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- at the end of the process making recominant DNA, there will be three types of bacteria:
- bacteria that have not taken up any plasmids
- bacteria that have taken up plasmids that do not contain the gene
- bacteria that have taken up recombinant plasmids
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what are some problems and difficulties with gene therapy?
- may introduce other genes
- may activate oncogenes which can cause cancers
- not permanent and therefore re-treatments are needed
- it is difficult to get the new functional genes into the genome of the cells
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what is a concern with using a virus as vectors to introduce the genes into cells?
- they could cause diseases
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what are some examples of things that could be used as vectors to introduce the genes into cells?
- liposomes
- plasmids
- viruses
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what is one way of getting liposomes into the body?
- to inhale the droplets in an aerosol like using an asthma inhaler
- the liposomes easily fuse with membranes introducing the genes into the cells
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what is the main sign of muscular dystrophy?
- progressive muscle weakness
(signs that typically appear between ages 2-3 include:
- frequent falls
- trouble running and jumping
- walking on toes
- difficulty getting up from a lying or sitting position
- muscle pain and stiffness
- learning disabilities
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why are boys more likely to suffer from muscular dystrophy?
- it is X linked, and boys only have one X chromosome
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is exon skipping a cure or treatment?
treatment
- if proven to be effective, this treatment would need to be repeated regularly
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what is a totipotent cell?
a cell that has the potential to develop into any type of cell
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the scaffolds in tissue engineering are artificial strictures that can support 3D tissues. what must it be able to do?
- allow cells to attach and move
- deliver and retain cells and biological molecules
- are porous to allow diffusion of nutrients and waste products
- are biodegradable and absorbed by the surrounding tissues
