application to genetics Flashcards
1
Q
when did the human genome project begin and how long did it take
A
The Human Genome Project began in 1990 and took ten years to complete,
but the analysis of all the sequences obtained took much longer.
2
Q
what were the aims of the human genome project
A
- Identify all the genes in the human genome and identify their loci
(positions on the chromosome). - Determine the sequence of the 3.6 billion bases present in the human
genome and store in databases. - Consider the ethical, social and legal issues that arise from storing this
information.
3
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).
4
Q
restriction enzymes
A
bacterial enzymes that
cut DNA at specifi c base
sequences.
5
Q
what is the polymerase chain reaction
A
a technique that produces a large number of copies of specifi c fragments
of DNA, rapidly.
6
Q
electrophoresis
A
a
technique that separates
molecules according to size.
7
Q
what was the method that the human genome project use
A
sanger sequencing
8
Q
what is sanger sequencing
A
- It works by sequencing small fragments of DNA around 800 bases in length created by the use of restriction enzymes.
- DNA polymerase was then used to synthesise complementary strands using
the polymerase chain reaction. - Four reactions were carried out (one for
adenine, thymine, cytosine and guanine), 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 these were incorporated into the complementary strand, further synthesis
was prevented.
9
Q
describe the difference between the sanger method and NGS
A
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.
10
Q
what is ngs
A
next generation sequencing
11
Q
what is the 100k genome project
A
Launched in 2012 using NGS, the project aims to sequence 100,000 genomes
from healthy individuals and patients with medical conditions across the
UK to establish any variance in their base sequences and identify if there is
a genetic correlation.
12
Q
what does the 100k project aim to do
A
It is hoped that diseases will be better understood and new treatments can be found.
13
Q
what are ethical concerns of the human genome and 100k projects
A
■ If a patient has 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 identi ed, 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 say for Alzheimer’s disease?
■ Could screening of embryos be extended from genetic diseases to desirable
traits?
■ How to ensure safe storage of patient data.
14
Q
how have we been closley related to primates
A
Other organisms have had their genomes sequenced, which has allowed
us to determine evolutionary relationships, e.g. how closely related we
are to primates.
15
Q
what is the mosquito responsible for
A
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.
16
Q
what is the mosquito genome project
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 won’t be released into the wild, it is an
exciting step forward in the control of malaria.
17
Q
what have been attempts of controlling malaria
A
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, e.g.
atovaquone, larium, artimesinin, but it is hoped that the sequencing of its
genome will allow for the development of new drugs.
18
Q
primer
A
a short single strand of DNA between
6 and 25 bases long that is complementary to the base sequence at one end of a singled-stranded DNA
template, acting as a start point for DNA polymerase to attach.
19
Q
what does PCR do
A
Using the polymerase chain reaction (PCR) technique, a large number of
copies of specific fragments of DNA may be made rapidly. From each strand
of DNA it is possible to produce over a billion copies in a few hours.
20
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)
which act as a start point for the DNA polymerase, and are complementary
to the start point on the DNA strand of interest.
■ Deoxyribonucleotides containing the four different bases.
■ A buffer.
21
Q
describe the steps of PCR
A
During the reaction, a thermocycler is used to rapidly change the temperature.
1. Step 1 – heat to 95oC to separate the DNA strands by breaking the
hydrogen bonds between the two complementary DNA strands.
2. Step 2 – cool to 50–60oC to allow the primers to attach by complementary base pairing (annealing).
3. Step 3 – heat to 70oC to allow the DNA polymerase to join complementary
nucleotides (extension).
4. Repeat 30–40 times.
22
Q
what are the limitations of PCR
A
■ Any contamination is quickly ampli ed (copied).
■ 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
concentrations of reagents reduce, and product builds up.
23
Q
short tandem repeats
A
short sections of DNA found in the non-
coding regions of the genome that show great variability in the number of times that they repeat from individual to individual, so can be used to produce a
genetic fingerprint.
24
Q
how can a genetic fingerprint be produced
A
- use the many variable regions of DNA which did not code for amino acids called short tandem repeats (STR) regions,
to produce a genetic fingerprint. - These regions were called microsatellites and there are thousands of them scattered throughout the chromosomes.
- The number of times that these regions are repeated gives individuality. PCR
is then used to amplify specific microsatellite sequences from very small
samples of DNA left at a crime scene. - Currently ten different microsatellite sequences are used to build up a unique ngerprint in UK (13 in US). When these different-sized fragments are visualised by
gel electrophoresis a unique banding pattern is created. - To visualise DNA, ethidium bromide is often used as it intercalates with DNA (inserts between the base pairs) and uoresces under ultraviolet light.
25
probe
short piece of DNA that is labelled with a
fluorescent or radioactive marker, used to detect the presence of a specific base
sequence in another piece of DNA, by complementary base pairing.
26
DNA hybridisation
single-stranded DNA
molecules anneal to
complementary DNA.
27
what is gel electrophoresis
Gel electrophoresis is a method of separating DNA fragments according to size.
28
describe the steps of gel electropheresis
* The gel used is made from agarose (a polysaccharide which is the main
constituent of agar) which contains pores in its matrix. DNA samples are
loaded 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.
* Size of fragments can be estimated if a sample of known DNA sized fragments (called a DNA ladder) is run at the same times as the samples.
29
what do DNA probes and how do they do gel electropheresis
* DNA probes can also be used to nd DNA sequences of interest within
DNA fragments.
* Probes, which are portions of single stranded DNA incorporating a radioactive tracer (32P) or fluorescent label, are designed to be complementary to part of the sequence of interest.
* When the probe is washed over the gel, it binds to the exposed complementary nucleotides by a process known as DNA hybridisation.
* The DNA fragment which contains
the sequence of interest is then identi ed by its fluorescence or radioactive
signal.
* To 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.
30
What is DNA profiling
* DNA profiling is a non-invasive procedure requiring hair samples, or a mouth swab to collect enough DNA which can be further ampli ed by PCR.
* It has been used successfully to provide evidence in criminal cases, as well as a
number of different situations:
31
what situations have DNA profiling been used for
■ To provide forensic evidence to identify or rule out suspects, or to identify
human remains.
■ To prove paternity, or in rare cases maternity. Here the genetic ngerprint
of the child is composed of elements of the ngerprints of both parents. It
has also been used to identify siblings.
■ In immigration applications where the right to remain in a country exists
for a parent and their children.
■ Phylogenetic studies where relatedness of species can be investigated to
suggest evolutionary links.
32
what are ethical and legal concerns with DNA profiling
* 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.
33
recombinant DNA
DNA
produced by combining
DNA from two different
species.
34
transgenic
an organism
that has been genetically
modifi ed by the addition of a
gene or genes from another
species.
35
what is genetic engineering
Genetic engineering allows genes to be manipulated, altered or transferred
from one organism or species to another, making a genetically modi ed
organism (GMO).
36
describe an example of genetic engineering
* It 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).
37
what is a transgenic organism
When the genetic material from two different species is combined, the
result is recombinant DNA, and if donor DNA is inserted into another
organism, the organism becomes transgenic.
38
what are the main tools in genetic engineering
restriction enzymes
39
what are restriction enzymes
* Restriction enzymes (restriction endonucleases) are bacterial enzymes that cut up any foreign DNA which enters a cell. When a restriction enzyme
makes a cut, the cut may be staggered and there are short, single-stranded
fragments at either end. These overhangs are called ‘sticky ends’. Sometimes
these enzymes cut DNA between specific base sequences which the enzyme
recognises and do not leave a ‘sticky end’. These are called ‘blunt cutters’:
40
describe how DNA is amplified and how the DNA of interest is found when inserting it into a plasmid
* Before the introduction of PCR, DNA could be amplified by inserting it into a
bacterial plasmid.
* When the bacterium containing the plasmid divides, the plasmid (and its inserted DNA) is copied.
* 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 are 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.
41
describe the process of using a restriction enzyme to insert a gene into a plasmid
1. The bacterial plasmid contains two
marker genes: the first one is for
ampicillin resistance so any bacteria
that contain the plasmid can grow
on an agar plate with ampicillin
on it, and their growth is used to
con rm 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
con rm insertion of target gene.
2. The plasmid is cut with a
restriction enzyme to open the
plasmid.
3. The foreign DNA or gene is cut with
the same restriction enzyme to
ensure complementary sticky ends.
4. DNA is inserted using DNA ligase
enzyme which joins the sugar-
phosphate backbones of the two
sections of DNA together.
5. To ensure that bacteria have a
plasmid with the donor gene in, the
second marker gene is used, e.g. Lac
Z gene. The Lac Z gene metabolises
x-gal turning it from colourless
to blue. Plasmids with an inactive
Lac Z gene (and hence containing
inserted DNA) will appear blue if
x-gal is spread on the plate as they
are unable to metabolise it.
42
DNA ligase
a bacterial enzyme that joins sugar-
phosphate backbones of two molecules of DNA together.
43
reverse transcriptase
an
enzyme that produces DNA
from a RNA template.
44
how is reverse transcriptase used
* Many eukaryotic genes contain introns, and are dif cult to locate within
3.6 billion bases spread across 46 chromosomes.
* To solve this problem,
scientists turned to another enzyme, reverse transcriptase which produces
complementary or copy DNA (cDNA) from a mRNA template.
* By targeting β cells in the pancreas, there is a high proportion of mature mRNA that codes for insulin, which can be extracted and reverse transcribed to cDNA.
* To ensure proper expression in bacteria, the human regulator sequence (which controls gene expression) is replaced by a bacterial regulator and the cDNA inserted into the plasmid using restriction enzymes and ligase as shown
below.
Once expressed in bacterial cells, the insulin can be puri ed for use.
45
what are advantages of genetically engineering bacteria
■ Allows production of complex proteins or peptides which cannot be made
by other methods.
■ Production of medicinal products, e.g. human insulin, factor VIII clotting
factor. These are far safer than using hormones extracted from other
animals or from donors. Many people with haemophilia within the UK
were infected with HIV during the 1980s from contaminated factor VIII
extracts.
■ 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.
46
what are the disadvatnages of genetically engineered bacteria
■ It is technically complicated and therefore very expensive on an industrial
scale.
■ There are dif culties 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 enzyme produces millions of
fragments which are of no use.
■ Not all eukaryote genes will express themselves in prokaryote cells.
47
what are hazards of genetically engineering 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 transfering oncogenes by using human DNA fragments
thus increasing cancer risks.
48
what are GM Crops
* The use of GM crops is widespread in the USA and is expanding in the EU,
Brazil, India and other countries.
* The advantages and disadvantages of eachGM crop are not fully proven.
* The most widely grown genetically modified crop is soya.
49
what are examples of GM crops
■ Insect-resistant crops using a gene which codes for a toxin from the
bacteria Bacillus thuringiensis (Bt).
■ Crops tolerant to herbicides like glyphosate (RoundupTM) or glufosinate
ammonium (LibertyTM).
■ Crops with stacked traits of both Bt insect resistance and herbicide
tolerance.
■ Virus resistant crops.
50
what are benefits of GM crops
■ Increased growth rates, e.g. high crop yields have been reported for cotton
and soya, but may not have been sustained.
■ Improved nutritional value, e.g. high vitamin A precursor content in Golden
Rice.
■ Increased pest resistance, e.g. Bt-maize.
■ 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.
51
what are concerns with GM crops
■ Genetic contamination, e.g. pollen from GM crops being transferred
to other crops which can lead to the development of superweeds e.g.
glufosinate – resistant oilseed range crossbred with the weed Charlock to
produce a herbicide-resistant weed.
■ Misuse of pesticides, e.g. overuse of the weed killer roundup on
RoundupTM-ready soya.
■ Control of agriculture, by corporations, e.g. the control of seed supplies to
farmers.
52
what are 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.
53
How are plants transformd with Agrobacterium tumifaciens
* Plasmid extracted from the A. tumifaciens.Restriction enzyme is used to cut the plasmidand remove the tumour-forming gene.
* The gene is inserted into the plasmid,
replacing the tumour-forming gene.
DNA ligase is used to join the donor
and vector DNA together.
* The plasmid is inserted
back into the bacterium.
* A section of DNA containing a gene
for disease resistance is located and
isolated using the same restriction
endonuclease.
* 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.
54
describe types of genetic diseases
Genetic diseases involve single gene conditions, e.g. Duchenne muscular
dystrophy and cystic fibrosis, chromosomal disorders, e.g. Down’s, and
multifactorial conditions where faulty genes are part of the cause, e.g.
Alzheimer’s disease.
55
what is genetic screening
* Screening for genetic conditions allows 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, and can help
families to plan to avoid passing on conditions to children, e.g. Tay-Sachs
disease, which is prevalent in Ashkenazi Jews.
* Screening can involve testing
parents, IVF embryos prior to implantation, foetuses during pregnancy
(pre-natal testing) and newborns.
* Screening involves sessions with a genetic counsellor so that the implications are fully understood.
* 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.
56
what is the aim of genetic therapy
* The aim of gene therapy is to treat a genetic disease by replacing defective
* alleles in a patient with copies of a new DNA sequence, but treatment can
also involve replicating the function of genes using drugs.
57
what are the two possible method of genetic therapy
* somatic cell therapy
* germ line therapy
58
what is somatic cell therapy
* the therapeutic genes are transferred into thesomatic (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. For example, in the use of liposomes containing copies of the normal allele, to treat cystic fibrosis.
59
what is germ line therapy
* sperm or eggs are modi ed by the introduction of functional genes, which are integrated into their genomes.
* This wouldallow the therapy to be heritable and passed on to later generations.
* This is rare due to ethical and technical reasons.
60
what is duchenne muscular dystrophy and what are they symptoms
* Duchenne muscular dystrophy (DMD) is a recessive, sex-linked form of muscular dystrophy affecting up to one in 3500 live male births.
* It is caused by
a mutation in the dystrophin gene resulting in the failure to produce dystrophin, an important structural component of muscle tissue.
* The result is severe wasting
of the muscles and sufferers are often wheelchair bound by the time they reach
teenage years, and life expectancy is only 27.
61
what is a treatment for DMD
* A drug called drisapersen has been
developed which aims to treat DMD by introducing a ‘molecular patch’ over
the exon with the mutation making the gene readable again.
* A shorter form of dystrophin is produced, but one thought to be more functional than the untreated version.
* This type of treatment is called exon skipping.
62
what is cystic fibrosis and symptoms
* Cystic fibrosis is caused by a recessive allele that codes for a mutated form
of the cystic brosis 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 brosis 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.
63
what is a possible treatment for cystic fibrosis
* Gene therapy has involved 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 be repeated.
64
describe the effectivness of gene therapy
* Results are variable, and therapy often has to be repeated, so it is 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.
65
what is genomics
Genomics is the study of the structure, function, evolution and mapping
of genomes, e.g. the Human Genome and 100K Projects.
66
how does genomics enable healthcare to be improved by
■ More accurate diagnosis of disease.
■ Better prediction of the effect of drugs and improved design of drugs.
Individual patients metabolise drugs in different ways, so it is 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.
67
why is NGS technology an advantage
* 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.
68
describe the process of tissue engineering
The first licensed engineered tissue in 1998 was an artificial skin called
‘Apligraf’ used in place of skin grafts for burns 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 mortality.
* 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.
* Tissue culture is used to grow large numbers of genetically identical cells
quickly from a single parent cell, and is referred to as therapeutic cloning.
* The main advantage of this is that if patient’s cells are used, rejection of the tissue is unlikely.
* During the tissue culture process, adequate oxygen and nutrients
must be provided, and optimal conditions, e.g. temperature and humidity
must be maintained.
* Reproductive cloning of humans involving whole organisms is prevented by law in the UK.
69
stem cell
an undifferentiated cell capable
of dividing to give rise to
daughter cells which can
develop into different types
of specialised cell or remain
as undifferentiated stem
cells.
70
what are stem cell
Stem cells are undifferentiated cells which can develop into numerous
different cell types given the correct trigger.
71
what are the main sources of stem cells
■ From embryos (embryonic stem cells).
■ Adult stem cells, e.g. bone marrow which 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.
72
what are the possible treatments of using stem cells
* Stem cells can be used to 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 burns victims.
* They can also be used to screen
new drugs, and develop model systems to study growth and birth defects.
73
what are advantages of stem cells
* Can be produced quickly.
■ Produced on a large scale.
■ Production of genetically identical cells for transplant, reducing risk of
rejection.
74
what are disadvantages of using 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.
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