Application and Flashcards
1
Q
What is the intended purpose of the Human Genome Project and the 100K Genome Project?
A
improve knowledge and understanding of genetic disorders and improve their diagnosis and treatment.
2
Q
When did the human genome project begin and complete?
A
began in 1990 and was completed in 2003
3
Q
What did the human genome project discover?
A
•The order of DNA bases in genomes.
•The distribution and sequences of introns and exons.
•The loci of individual genes.
4
Q
What did scientists do in the human genome project?
A
-used ‘Sanger Sequencing’, a DNA sequencing process that involved reading the base sequence of a length of DNA.
-This method sequenced relatively small sections of DNA at a time (usually <1,000 bps) and took a long time.
-For example, it took a year to sequence a million base pairs.
5
Q
What are there now and what are these called?
A
many faster techniques in use that can sequence the entire genome (all of the genetic information of an organism) in a few hours.
These new rapid techniques, collectively known as Next Generation Sequencing (NGS), enable scientists to study variation within the human genome.
6
Q
What is the 100k genome project?
A
was launched in 2012.
It used NGS to sequence 100,000 genomes from NHS patients with cancer or rare diseases.
7
Q
What did the 100K genome project project enable us to do?
A
•Locate genes responsible for rare diseases and cancers
•Locate mutated genes
•Compare genomes of genetic disorder sufferers with the normal genome
•Develop treatments for genetic disorders
•Undergo effective prenatal diagnosis for genetic disorders
8
Q
What has identification of allele sequences enabled scientists to do?
A
to scan a patient’s DNA sample for mutated sequences and to compare the sequence of DNA bases in a patient’s gene to a normal version of the gene
9
Q
What can IVF embryos have done?
A
be screened for the presence of alleles which cause conditions including cystic fibrosis, Huntington’s disease and thalassaemia.
10
Q
where else can genetic screening be useful?
A
in association with genetic counselling, allowing a couple to make informed decisions before having children
11
Q
What has been produced by the projects and what does this mean?
A
A vast quantity of data has been produced
- We do not know how this information might be used in the future.
Society has yet to decide how it should be treated and where legal and moral responsibilities lie.
12
Q
What are Ethical issues regarding the screening of DNA?
A
-Ownership of genetic information that could lead to potential discrimination e.g. health insurance, job applications, social stigmatisation and misuse of the data.
-There are a number of concerns regarding the possibility of routine screening for adult-onset disorders such as Alzheimer’s disease and some cancers. Some people do not want to learn this information about themselves, and it could cause anxiety.
-Concerns have arisen over embryo screening and the potential for choosing alleles to ensure specific characteristics: ‘designer babies’.
13
Q
What is one of the case studies?
A
Screening of embryos for cystic fibrosis
14
Q
What are sufferers with cystic fibrosis?
A
homozygous for an autosomal recessive allele and lack a functioning CFTR gene
15
Q
What happens in cystic fibrosis?
A
Mucus blocks the airways and results in symptoms such as difficulty breathing, coughing and recurrent chest infections
16
Q
What is the effect of inheriting cystic fibrosis?
A
•Cl- can’t be transported out of epithelial cells.
•Water potential outside cells doesn’t lower and so water doesn’t leave cells by osmosis.
•This means mucus is thick and sticky and accumulates.
17
Q
What none humans have genome’s been sequenced for and what has this allowed?
A
-mosquito, Anopheles gambiae and the Plasmodium parasite, as well as chimpanzees and other primates. -This has allowed scientists to look at evolutionary relationships and to conserve species in the future.
18
Q
What are two other methods that could be used to determine how closely related organisms are?
A
DNA hybridisation and amino acid sequence analysis
19
Q
Where is malaria common and what have been used to control it?
A
-common in tropical and subtropical regions around the equator. -chemicals have been used to attack both the vector, Anopheles gambiae, and the parasite, Plasmodium sp. in an effort to control the spread of this disease.
20
Q
What is hampering attempts to irradiate malaria (which is responsible for over one million deaths a year)? (control of the vector)
A
Rapid evolution of insecticide resistance in the Anopheles gambiae mosquito
21
Q
What resistance is a problem and why?
A
Pyrethroid resistance is a problem as this is the only insecticide safe for use with the nets that people sleep under.
22
Q
When was the DNA sequence of the Anopheles gambiae genome was completed and what is it allowing scientist to do?
A
allowing scientists to develop chemicals that could render the mosquito susceptible to insecticides again, preventing it from transmitting malaria.
23
Q
What has plasmodium sp also developed?
A
multi-drug resistance
24
Q
When was the genome of Plasmodium sp sequenced and what is hoped
A
-in 2002.
-It is hoped that a better understanding of genetic control of Plasmodium infection will allow the development of more effective drugs.
25
Give two advantages and two disadvantages of the chemical control (pesticide) of
mosquitoes.
ADY.quick and easy to apply chemicals; fast results; application does-not require a high level of skill
DIS - fear of resistance, can kill beneficial organisms
26
Using your knowledges of natural selection, describe how the mosquito population in africa became resistant to insecticides like thyroid?
-Selection pressure of insecticide meant individuals with the resistant
allele had a selective advantage
-These mosquitos survive and reproduce, passing on the resistance allele to'the next generation.
-Resistance allele increases in frequency within the population.
27
Synthesis of antibodies can protect mosquitoes against Plasmodium. Describe the structure of an antibody. (3)
-Antibodies are globular proteins, they have a quaternary structure which means they have a specific 3D shape.
-They contain peptide bonds between amino acids and ionic, disulphide and hydrogen bonds between R-groups.
28
1.Suggest how the synthesis of these antibodies in mosquitoes could help to reduce malaria outbreaks in humans in the future? (2)
•The mosquito is able to produce antibodies to combat the plasmodium parasite.
•The plasmodium could no longer survive in the mosquito.
•The mosquito would no longer spread infection when biting people in the future.
29
What does PCR do?
•allows the quantity of DNA to be amplified rapidly for analysis.
•At a crime scene a small sample of DNA may be found.
•In order to carry out tests, larger samples of DNA are required.
•Using the PCR technique copies of specific fragments of DNA may be made.
30
what are the three stages of PCR?
Separation
Annealing
Extension
31
What happens in separation?
The target DNA molecule is dissolved in a buffer and is heated to 95ᵒC, this denatures the DNA causing the two stands to separate, exposing the nitrogenous bases ON BOTH STRANDS (hydrogen bonds broken between complimentary strands)
32
what happens in annealing?
•The sample is cooled to 50-60ᵒC to allow the short DNA primers to bind to the DNA strands
•They form hydrogen bonds with complementary bases.
33
What does a primer do? (annealing)
A primer must anneal to the start of the DNA strand to then allow Taq polymerase to bind;
34
why are two different primers needed?
2 different primers are needed as they anneal to different strands
35
what happens in extension?
•Heating to 70ᵒC allows thermally stable DNA polymerase (Taq polymerase) to add complementary nucleotides
•It forms phosphodiester bonds in the sugar phosphate backbone, this creates 2 double stranded molecules
36
How do you work out how many dna molecules have been made?
2 to the power of n (n=number of cycles)
37
what happens at the end of the PCR?
-The cycle is repeated.
-40 cycles can produce over 1 billion copies of the target sequence from just one piece of DNA.
38
39
At the end of one cycle, how many molecules of DNA have been produced from each original strand?
•Forensics = amplification of small quantities of DNA in a blood sample
•Synthesis of a small gene sequence for insertion into a plasmid (by DNA ligase)
40
what is meant by the term primer and what does it do?
Single stranded molecule of DNA
•Usually 10-20 nucleotides (nitrogenous bases) long
•Binds by hydrogen bonding (anneals) to a complementary DNA base sequence (template)
•Required for activity of (Thermostable) DNA polymerase - the polymerase needs DNA as its substrate to build new strand from
41
Which enzyme is required at step 3?
Thermostable (Taq) DNA polymerase
42
The enzyme required at step 3 is described as thermophilic. Explain what thermophilic means and why this is important for the process of PCR?
•Enzyme is stable even at very high temp So the enzyme is not denatured at this high temp;
•Important in PCR as the temperature is increased to 95 0 C and never usually falls below 55 0 C, this temperature is too high for activity of most enzymes found in nature.
43
What are limitations of PCR?
•error rate
•limits on amplification
•DNA fragment size
•Inhibitors
•Contamination
•Sensitivity
44
what is the limitation of contamination?
Any DNA that enters the system by mistake will be amplified.
Sources may be air-borne or come from the previous PCR reactions using the same apparatus.
45
What is the limitation of error rate?
Taq polymerase can not proofread and correct errors in the base sequence as it is a bacterial DNA polymerase.
Each cycle copies and multiplies DNA, so these errors accumulate.
46
what is the limitation of sensitivity to inhibitors?
Molecules in the sample may act as inhibitors and PCR is very sensitive to them, e.g.
-Phenolics, especially in plant material
-Humic acids is archeogical specimens
These act as inhibitors by either interacting with DNA or interfering with thermostable DNA polymerase.
47
What is the limitation of limits on amplification?
After about 20 cycles PCR slows down and plateaus because:
-DNA primers and DNA nucleotide concentrations {run out / become limiting}
-The enzyme denatures after repeated heating
-DNA in high concentration causes the single stranded molecules to base pair with each other rather than with primers.
48
What is the limitation of DNA fragment size?
PCR is most efficient at making DNA about 1000-3000 base pairs long because taq polymerase can not correct its errors.
Many genes, including human genes, are much longer than this
49
look in booklet.
50
what is gel electrophoresis?
a method of separating DNA fragments according to size to produce a genetic fingerprint
51
What is the gel made from?
agarose, similar to agar, which contains pores in its matrix
52
What happens before gel electrophoresis?
Each DNA sample is first treated with a restriction endonuclease enzyme, to cut the DNA into smaller fragments…
2.Restriction endonucleases hydrolyse phosphodiester bonds cutting the sugar-phosphate backbones…
3.They recognise specific base sequences in the DNA for cutting..
53
What is the firsts step of gel electrophoresis?
DNA samples are loaded into wells at one end and a voltage is applied across the gel
54
what are the uses of DNA profiling in society?
Twins - determining whether babies are monozygotic or fraternal twins
Siblings - people who have been adopted may wish to determine blood relatives
• Phylogenetic studies to determine relatedness
Paternity testing
• Immigration
• Forensic use
55
What is the last stage of gel electrophoresis?
-DNA ladder can be run alongside the sample.
-A DNA ladder contains DNA fragments of known length. This can be used to determine the length of DNA in the sample being analysed.
56
what are Exons?
regions of DNA that code for proteins.
57
What are between Exons?
regions of non-coding DNA called introns, which contain blocks of repeated nucleotides.
These repeating blocks of DNA bases are called short tandem repeats (STRs) and do not code for proteins.
58
In the UK, what are STRs used to do?
build up a unique fingerprint.
59
why do we separate intron when producing a DNA profile by gel electrophoresis??
as there are far more differences between the introns of individuals than between exons
60
What is the definition of short tandem repeats?
shorthand and repeats are repeating blocks of DNA bases
61
what are the uses of DNA profiling in society?
•Twins - determining whether babies are monozygotic or fraternal twins
•Siblings - people who have been adopted may wish to determine blood relatives
•Phylogenetic studies to determine relatedness
•Paternity testing
•Immigration
•Forensic use
62
What does genetic engineering do?
allows DNA to be manipulated, altered or transferred from one form to another = foreign DNA can be inserted into the DNA of a different species so it can make that foreign protein
63
What are the application applications of genetic engineering?
1.Transfer genes into bacteria to make useful products such as human insulin
2.Transfer genes into plants so they acquire new characteristics e.g. disease resistance
3.Transfer genes into humans so they no longer suffer from genetic diseases e.g. defective allele replaces: Cystic fibrosis
64
What stages does the process of a new protein using DNA technology of a gene transfer include?
1.Identify and isolate DNA fragments from donor organism using restriction enzymes or reverse transcriptase
2. Insert DNA fragments into a vector (recombinant DNA)
3. Transfer the recombinant DNA into a suitable host cell
4. Identify host cells which have taken up and are expressing the recombinant DNA.
5. Clone the host cells
65
what is donor DNA?
- A DNA fragment containing a desired gene from a donor individual
66
What is a vector?
Used to transfer the donor DNA into the suitable host cell
67
What is recombinant DNA?
Produced when the donor
DNA fragment is spliced into the DNA of another organism
68
What is a clone?
Genetically identical copy
69
What is the first step to the formation of recombinant DNA?
identify and isolate DNA
70
What are the two ways the desired gene can be isolated?
•Locate it on the donor DNA and cut out using restriction endonuclease (enzymes that cut DNA as specific base sequences).
•Extract mRNA from a cell actively synthesising the required protein/ polypeptide and use reverse transcriptase and DNA polymerase to produce a double stranded cDNA fragment.
71
What is the explanation of using reverse transcriptase?
Isolating a gene can be difficult. However, it is likely that there will be large numbers of mRNA molecules in the cytoplasm of the cell which will have been transcribed from this gene.
72
what can reverse transcriptase be used to do? (2)
to produce a single strand of DNA from this mRNA
73
What can DNA polymerase enzymes then do?
can then add free DNA nucleotides to the template strand to produce a double stranded section of cDNA which will code for the desired protein.
74
look in book
75
how is cDNA produced?
1.mRNA is extracted from donor cells e.g. from beta cells in the pancreas (for the gene for insulin production).
2.Reverse transcriptase enzymes are used to make a DNA copy of the mRNA.
4.DNA polymerase enzymes catalyse the addition of free DNA nucleotides. This converts the cDNA into a double strand.
5.DNA is copied many times using the PCR.
76
What are the benefits of the use of reverse transcripts to produce cDNA?
•Overcomes the problem of locating the gene on DNA.
•Avoids restriction enzymes cutting the desired gene into non-functional fragments.
•No introns present in cDNA.
•No need for post-transcriptional processing to produce functional mRNA.
77
What is the second step of the formation of recombinant DNA?
inserting the DNA into a vector
78
What is a common vector used in genetic engineering
a plasmid- these plasmids are isolated from bacteria
79
What is a plasmid?
Small rings of circular DNA found in the cytoplasma of bacterial cells
80
What are the steps of inserting the DNA into a vector?
1.Cut the isolated donor DNA using restriction endonuclease (restriction enzymes). These cut the DNA between specific base sequences (restriction sites), such as GAATTC.
Many restriction enzymes cut DNA in a staggered pattern and leave overhanging ends with unpaired bases exposed. These are known as “sticky ends”.
2. The same restriction enzymes are then used to cut the plasmid DNA at the same base sequences.
3. Another group of enzymes, NA ligase enzymes, are used to splice goin) together the sugar-phosphate backbones of the donor and vector DNA.
4.The new plasmid is now known as recombinant DNA.
81
Why should the same restriction enzyme be used on the DNA isolated for insertion and the vector (plasmid) DNA?
To ensure.theunpaired.bases.(sticky.ends).on the.donor.DNA
•fragment and plasmid are complementary
This means complementary bases can form hydrogen bonds
82
What is the first step of the formation of recombinant DNA?
Transfer of recombinant DNA into a host cell-
•These recombinant plasmids are mixed with bacterial cells and calcium chloride in the hope that bacteria will take up the plasmid. •However, often as few as 1% of the bacterial cells take up the plasmid and become transformed.
83
What is the fourth step to the formation of recombinant DNA?
Identify host cells which have taken up the recombinant DNA and are expressing it
84
what do you scientists use a marker gene to do?
To identify the bacterial cells which have taken up the plasmid and therefore have the desired gene
85
What is the marker usually?
usually a gene which confers antibiotic resistance.
86
What will cells that take up the desired gene also do?
will also take up the antibiotic resistant gene.
Therefore, if they are grown on a medium containing antibiotics, only the cells containing the plasmids will survive.
87
What is an example of marker gene?
The human gene for blood clotting was inserted into bacterial plasmids. Some plasmids did not take up the human gene.
Replica plating was used to identify the bacteria with the human gene
88
check book.
89
What is the 5th step of the formation of recombinant genes?
the bacteria can be grown on a large scale to produce large amounts of for eg insulin
90
What are the pros of genetic engineering of bacteria?
-Allows the manufacture of medical productions e.g. insulin to treat diabetes and human clotting factors to treat haemophilia
-Prevention and treatment of disease e.g. produce vaccines.
-Enhancing crop growth – modified bacteria secrete molecules toxic to pests.
91
What are the cons of genetic engineering of bacteria?
-Plasmids are easily transferred. Genes may be exchanged with other bacteria and antibiotic resistance marker genes could be transferred to pathogenic bacteria
-A new microorganism with a new gene may become a threat if released into the environment
-The possible transfer/activation of oncogenes by using fragments of human DNA
92
What are genetically modified soy beans?
-Important source of food.
-Used to produce a wide range of products e.g. flour, protein, oil,
bread and soya milk. ‘Roundup ready’ soybeans are genetically
modified to contain genes for herbicide resistance.
-The crops can be sprayed to remove weeds without inhibiting their growth
93
94
What are Bt tomatoes?
Bacillus thuringiensis is a bacterium that lives in the soil.
It contains genes that produce a protein that acts as an
insecticide.
95
Suggest how transfer of this gene into tomato cells would be of benefit. (2)
Tomatoes not targeted by insects;
Less.crop.spoilage;
Higher crop yield
96
What are antisense tomatoes?
Tomatoes ripen naturally when they produce an enzyme
that breaks down their cell wall.
•A second copy of this gene was inserted into tomato plant
cells to prevent translation and block the production of this
enzyme.
97
Suggest the benefit of this gene transfer (2)
Less food spoilage during transport
Longer shelf life
98
What have transformed plants been suggested as a way to do?
as a way of increasing food supply, enhancing nutritional value and making crops disease resistant and drought tolerant.
99
What are the arguments for gm crops?
-Substantial reduction in pesticide use on crops engineered for resistance to fungal pathogens and insect attack.
-Superior keeping qualities.
-Higher crop yield.
100
What are the arguments against gm crops?
-Pollen from GM plants engineered for herbicide resistance might transfer genes to wild relatives.
-Number of crop varieties may become limited = reduction in biodiversity.
-It is claimed there may be adverse effects of eating a crop that is expressing a new gene, making a new protein.
101
What is gene therapy?
genetic diseases can be treated by replacing genes or replicating the function of genes using drugs.
102
What’s the definition of gene therapy?
Replacing defective alleles with copies of a new functional DNA sequence
103
What is the main aim of gene therapy?
to treat a genetic disease by replacing defective alleles in a patient with copies of a new DNA sequence.
104
What methods can be used in gene therapy to introduce DNA into target cells?
•A virus as a vector
• A plasmid as a vector
• Injection of naked plasmid DNA
105
What are the two ways of replacing defective genes?
-Somatic cell therapy.
Targets body cells in affected tissues
-Germ line therapy.
This very rare and introduces corrective genes into germ line cells so genetic corrections are inherited.
106
What are the problems with somatic cell therapy?
Genetic changes are not inherited and do not appear in future generations
107
Suggest why germ-line therapy can be controversial
Genes interact with one another. Influencing genes in the gamete has unpredictable effects in future generations
108
What is an application of gene therapy?
DMD (a recessive, sex linked form of muscular dystrophy. It affects up to 1 in 3500 live male births)
109
What is dystrophin?
-a structural protein found in muscle.
/People with DMD fail to produce dystrophin and consequently have severe muscle loss and often become wheelchair-bound by the time they are teenagers.
Life expectancy is only 27
110
Name the group of proteins that comprise part of muscles. State two structural features of these proteins.
Fibrous, repeating amino acid in primary structure, alpha helices, twisted, rope like structure
111
What are most cases of DMD caused by?
one or more deletions in the dystrophin gene.
These genes have 79 exons; deletions in any of these alter the reading frame of the dystrophin mRNA.
The ribosome meets a stop codon too soon and the full polypeptide is not translated.
Therefore, functional dystrophin is not synthesised.
112
What is the drug drisapersen?
an antisense oligonucleotide.
It is a sequence of 50 nucleotides that is complementary to the mutated sequence
113
What does drisapersen do?
-treats DMD by acting as a ‘molecular patch’, binding to the mRNA over the exon with the deletion.
-That portion of mRNA becomes double stranded, so the ribosome can’t translate it.
-This restores the reading frame, so that a shorter, partially functional dystrophin protein can be made. -This type of treatment is called exon skipping.
114
How is drisapersen delivered to the patient?
in subcutaneous (under the skin) injections
115
What are the disadvantages of gene therapy?
•Only a small proportion of the introduced genes are expressed.
•When using a virus as a vector there are a number of potential issues:
•There may be an immune response in the patient.
•The virus may invade non target host cells.
•The virus could potentially become pathogenic and cause disease.
•The virus may affect other genes such as formation of oncogenes
116
What is genomics?
is the study of the structure, function, evolution and mapping of genomes as exemplified by the Human Genome Project and the 100K projects.
117
What should genomics enable?
healthcare to be improved by:
•more accurate diagnosis.
•better prediction of the effect of drugs.
•improved design of drugs.
•new and improved treatments for disease.
118
With the introduction of NGS technology, what may it be possible to do?
to look at tailoring therapies to individual patients, where an individual could have a unique treatment for a common disease
119
What is tissue engineering and what is its goal?
Uses biochemistry, cell biology, engineering and material science to repair, improve or replace biological tissues. Its goal is to produce ‘off the shelf’ bio-artificial organs and to regenerate injured tissue in the body.
120
What are stem cells?
undifferentiated cells that have the ability to become many different specialised cell types e.g. meristems
121
What can stem cells be used to do?
used for replacing damaged tissues and organs during tissue engineering- when a stem cell divides by mitosis, each daughter cell can remain a stem cell or become another type of cell with a more specialised function.
122
Where are embryonic stem cells found?
in 3-5 day old embryos
123
Where are adult stem cells found and what do they do?
in some adult tissues like bone marrow.
They replace cells that are lost through wear and, injury or disease, but they cannot form all cell types.
124
What are some examples of tissues and/or organs that have been created using tissue engineering?
skin
epithelial cells
125
What do cells grown in tissue culture do?
form cell lines that are clones.
All the cells are derived from a single parent cell and are genetically identical.
These cell lines can then be used to produce cloned tissue samples or potentially organs.
126
What is the role of stem cells in tissue engineering?
•Use to regenerate tissues and organs.
•To screen new drugs.
•To develop model systems to study normal growth and identify the causes of birth defects.
•To investigate the events that occur during human development.
127
What are some requirements for the use of stem cells from embryos in the uk? (ethical issues)
-Stem cells left over from IVF are deposited in the UK stem cell bank so they are available for other research groups. Donors must give specific consent to embryos created with their gametes being used in stem cell research
-There is no financial reward for any development or discovery made using them.
128
What do people involved in research say the use of embryonic stem cells helps to do?
clarify biological mechanisms and that a pre-14 day old embryo is a ball of cells with no possibility of independent existence.
129
Discuss and the suggest the possible arguments against the use of stem cells.
•The moral status of an embryo. An embryo does have moral rights, but not to the same degree as a living person.
•Fear that the use of stem cells may lead to humans being cloned
130
What are ethical issues of cloning of human tissues and organs?
There is fear that stem cells may lead to humans being cloned, an act that fundamentally devalues human life.
131
