manipulating genomes Flashcards
1
Q
What is the genome of an
organism?
A
All of the genetical material • For eukaryotes, it is the DNA in the nucleus and the mitochondria • Only 2% of your total DNA codes for proteins (exons) • The large non-coding regions of DNA that are removed from mRNA before it is translated are called introns
2
Q
What is satellite DNA?
A
Short sequences of DNA that are
repeated many times within introns,
telomeres and centromeres
3
Q
What is a minisatellite?
A
A sequence of 20-50 base pairs that will be repeated from 50 to several hundred times • Occur at more than 1000 location in the human genome • Also known as variable number tandem repeats (VNTRs
4
Q
What is a microsatellite?
A
A region of 2-4 bases repeated only
5-15 times
• Also known as short tandem
repeats (STRs)
5
Q
What are the similarities and
differences in satellites in
different people?
A
Always appear in the same positions on the chromosomes • The number of repeats of each mini or microsatellite varies between individuals, as different lengths of repeats are inherited from both parents
6
Q
What is DNA profiling?
A
Producing an image of the patterns in the DNA of an individual • A technique employed by scientists to assist in the identification of individuals or familial relationships
7
Q
What is gel electrophoresis?
A
A technique used to separate cut fragments of DNA • Uses the way charged particles move through a gel medium under the influence of an electric current • The gel is then immersed in alkali in order to separate the DNA double strands into single strands • The single-stranded DNA fragments are then transferred onto a membrane by Southern blotting
8
Q
What are the stages in
producing a DNA profile?
A
1. Extracting the DNA DNA is extracted from a tissue sample. Using the polymerase chain reaction (PCR), the tiniest fragment of tissue can give scientists enough DNA to develop a profile 2. Digesting the sample The strands of DNA are cut into small fragments using restriction endonuclease enzymes. They are cut at a specific nucleotide sequence called a restriction/ recognition site. 2 cuts are made, one through each strand of the DNA double helix. 3. Separating the DNA fragments The cut fragments of DNA are separated to form a clear, pattern, using gel electrophoresis 4. Hybridisation Radioactive or fluorescent DNA probes are added in excess to the DNA fragments on the membrane. Theses are short DNA or RNA sequences complementary to a known DNA sequence. They bind to the complementary strands of DNA and identify the microsatellite regions 5. Seeing the evidence X-ray images can be taken if radioactive labels where added to the DNA probes, and UV can be used if fluorescent labels were added
9
Q
What is the Polymerase chain
reaction (PCR)?
A
A version of the natural process by
which DNA is replicated, and allows
scientists to produce a lot of DNA
from the tiniest original sample
10
Q
How is the PCR machine used?
A
(aka a thermal cycler) • Temperature is carefully controlled and changes rapidly at programmed intervals • The reaction can be repeated many times by the PCR machine, which cycles through the programmed settings • 30 repeats gives around 1 billion copies of the original DNA sample, which is more than enough to carry out DNA profiling
11
Q
What are the steps involved in
the PCR?
A
1. Separating the strands • Temperature in the PCR machine is increased to 90-95°C for 30 seconds • This denatures DNA by breaking the hydrogen bonds holding the DNA strands together so they separate 2. Annealing of the primers • Temperature is decreased to 55-60°C and the primers bind (anneal) to the ends of the DNA strands • They are needed for the replication of the strands to occur 3. Synthesis of DNA • Temperature is increased to 72-75°C for at least 1 minute, as this is the optimum temperature for DNA polymerase • DNA polymerase adds bases to the primer, building up complementary strands of DNA and so producing double-stranded DNA identical to the original sequence • The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria found in hot springs
12
Q
What are the uses of DNA
profiling?
A
• Forensic science and criminal investigations • Proving the paternity of a child or in immigration cases to prove or disprove family relationships • To identify the species to which an organism belongs and to demonstrate evolutionary relationships between different species • To identify individuals who are at risk of developing particular diseases
13
Q
Give a brief history of DNA
Sequencing
A
1970s • Sanger sequencing enabled Fredrick Sanger and his team to read sequences of 500-800 bases at a time • Technique involved radioactive labelling of bases and gel electrophoresis on a single gel 1990 • Human Genome Project was established (HGP) • Scientist from a number of countries worked to map the entire human genome, making the data freely available to scientist all over the world • The first complete human genome sequence was published in 2003
14
Q
How does DNA sequencing
work?
A
• DNA is chopped into fragments, and each fragment is sequenced • The process involves terminator bases which are modified versions of A, C, T and G, which stop DNA synthesis when they are included • An A terminator will stop DNA synthesis at the location that an A base would be added etc • A is green, G is yellow, T is red, C is blue
15
Q
Describe the sequencing
process (capillary method)
A
1. Same process as in DNA profiling to produce double-stranded DNA. Mix DNA with a primer, DNA polymerase, an excess of normal nucleotides, and terminator bases 2. When a terminator base is added instead of a normal nucleotide, the synthesis of DNA is terminated. This results in many DNA fragments of random different lengths. After many cycles, all of the possible DNA chains will be produced with reactions stopped at every base. The fragments are separated according to their length by capillary sequencing. Lasers detect the different colours of the fluorescent markers on the terminator bases and thus the order of the sequence. 3. The order of bases in the capillary tubes shows the sequence of the new, complementary strand of DNA, which is used to build up the sequence of the original DNA strand. The data is them fed into a computer that reassembles the genomes by comparing all the fragments and finding the areas of overlap between them
16
Q
Computer analysis of all data
to give original DNA sequence
A
17
Q
What is capillary sequencing?
A
• Works like gel electrophoresis in
minute capillary tubes
18
Q
Describe next-generation
sequencing
A
• Instead of using a gel or capillaries, the sequencing reaction takes place on a plastic slide called a flow cell • Millions of DNA fragments are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments • Sequencing process still uses a principle of adding a coloured terminator base to stop the reaction so an image can be taken • Known as ‘massively parallel sequencing’ as all of the clusters are being sequenced and imaged at the same time
19
Q
What is bioinformatics?
A
The development of the software and computing tools needed to organise and analyse raw biological data • Includes the development of algorithms, mathematical models, and statistical tests
20
Q
What is computational
biology?
A
The use of data from bioinformatics to build theoretical models of biological systems, which can be used to predict what will happen in different circumstances • The study of biology using computational techniques, especially in the analysis of huge amounts of biodata • Helps us use the information from DNA sequencing e.g. in identifying genes linked to specific diseases
21
Q
What are genome-wide
comparisons used for?
A
Analysing the human genome • Analysing the genomes of pathogens • Identifying species (DNA barcoding) • Searching for evolutionary relationships
22
Q
How is the human genome
analysed?
A
• Since the first complete draft of the human genome was published in 2003, research projects e.g. the 100,000 Genome Project, have sequenced human genomes • Computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vulnerable
23
Q
What does the analysis of the
genomes of pathogens allow?
A
Doctors to find out the source of an infections. e.g. bird flu or MRSA in hospitals • Doctors to identify antibioticresistant strains of bacteria, ensuring antibiotics are only used when they will be effective • Scientists to track the progress of an outbreak of a potentially serious disease and monitor potential epidemics, e.g. flue and Ebola • Scientists to identify regions in the genome of pathogens that may be useful targets in the development of new drugs, and to identify genetic markers for use in vaccines
24
Q
How is analysis used in
identifying species (DNA
barcoding)?
A
• Using traditional methods of observation, it can be very difficult to determine which species an organism belongs to, or if a new species has been discovered • In the International Barcode of Life (iBOL) project, scientists identify species using relatively short sections of DNA from a conserved region of the genome
25
Which region of DNA is used to
analyse the animal species,
and why?
```
• A 648 base-pair sectional the
mitochondrial DNA in the gene
cytochrome oxidase, that codes
for an enzyme involved in cellular
respiration
• The section is small enough to be
sequenced quickly and cheaply
• Varies enough to give clear
differences between species
```
26
Why isn’t the same region of
DNA used to analyse land
plants?
That region of DNA doesn’t evolve
quickly enough in land plants to
show clear differences between
species
27
What region of DNA is used to
| analyse land plant species?
Two regions in the DNA of
| chloroplasts are used
28
What are the drawbacks of the
| barcoding system?
Scientists have not come up with
suitable regions for fungi and
bacteria yet, and they may not be
able to do so
29
How are genomes used to
search for evolutionary
relationships?
```
DNA sequences of different
organisms can be compared
• The basic mutation rate of DNA
can be calculated and scientists
can calculate how long ago 2
species diverged from a common
ancestor
• DNA sequencing allows scientists
to build up evolutionary trees with
great accuracy
```
30
What is proteomics?
```
The study and amino acid
sequencing of an organisms entire
protein complement
• In the past scientists though each
gene codes for a different protein,
but we now know that there are
20-25,000 coding genes in human
DNA, and a different number of
unique proteins
• The DNA sequence of the genome
in theory should enable you to
predict the sequence of the amino
acids in all of the proteins it
produces, but some genes can
code for may different proteins
```
31
What happens to mRNA
transcribed from DNA in the
nucleus?
```
• Before it lines up on the ribosomes
to be translated, this ‘pre-mRNA’
is modified in a many ways
• Introns are removed, and in some
cases some of the exons are
removed as well
• The exons to be translated are
joined together by enzyme
complexes known as
spliceosomes
• Spliceosomes may join the same
exons in a variety of ways
• Therefore a single gene may
produce several versions of
functional mRNA, which in turn
would code for different
arrangements of amino acids ,
giving different proteins and
phenotypes
```
32
What happens in protein
| modification?
```
Some proteins are modified by
other proteins after they are
synthesised
• A proteins that is coded for by a
gene may be modified to give a
variety of other proteins
```
33
What is synthetic biology?
```
The design and construction of new
artificial biological pathways,
organisms or devices, or the
redesign of existing natural
biological systems
```
34
Give the different techniques
| included in synthetic biology
```
• Genetic engineering: a change in
a biological pathway, or relatively
major genetic modification of an
entire organism
• Use of biological systems in
industrial contexts: e.g. the use
of fixed or immobilised enzymes
and the production of drugs from
microorganisms
• Synthesis of new genes to
replace faulty genes: e.g. in
developing treatments for cystic
fibrosis, scientists attempted to
synthesis functional genes in the
lab and use them to replace faulty
genes in the cells of people
affected by CF
• Synthesis of an entire new
organism: in 2010, scientists
announced that they had created
an artificial genome for a
bacterium and successfully
replaced the original genome with
the new, functioning genome
```
35
What is genetic engineering
| also known as?
Recombinant DNA technology,
because it involves combining
DNA from different organisms
• Genetic modification
36
Give an overview of the stages
| needed in genetic engineering
1. The required gene is obtained
2. A copy of the gene is placed
inside a vector
3. The vector carries the gene into
a recipient cell
4. The recipient expresses the
novel gene
37
What are the methods for
| isolating the desired gene?
```
• The mRNA for the desired gene
can be isolated, and using the
enzyme reverse transcriptase, a
single strand of complementary
DNA can be made
• If scientists know the nucleotide
sequence of the gene, the gene
can be synthesised using an
automated polynucleotide
synthesiser
• If scientists know the sequence of
the gene, they can design PCR
primers to amplify the gene form
the genomic DNA
• A DNA probe can be used to
locate a gene within the genome
and the gene can then be cut out
using restriction endonuclease
enzymes
```
38
What are the benefits of using
| restriction endonucleases?
```
• Many restriction endonucleases
cut the 2 DNA strands unevenly,
leaving 1 of the strands a few
bases longer than the other strand
• These region with unpaired,
exposed bases are called ‘sticky
ends’
• These sticky ends make it easier
to insert the desired gene into the
DNA of a different organism
```
39
What is the advantage of
isolating mRNA and using
reverse transcriptase?
```
• It makes it easier to identify the
desired gene, as a particular cell
will make some very specific types
of mRNA
• e.g. ß cells fo the pancreas make
insulin, so produce lots of insulin
mRNA molecules
```
40
What are the most commonly
used vectors in genetic
engineering?
```
Bacterial plasmids
• Small circular molecules of DNA
separate from the chromosomal
DNA that can replicate
independently
• Once a plasmid gets into a new
host cell, it can combine with the
host DNA to form recombinant
DNA
```
41
What is a feature of plasmids
| chosen to be vectors?
```
• They contain a marker gene
• e.g. they may have been
engineered to have a gene for
antibiotic resistance
• This gene enables scientists to
determine that the bacteria have
taken up the plasmid, by growing
the bacteria in media containing
the antibiotic
```
42
How is DNA inserted into
| plasmids?
```
1. The same restriction
endonuclease as used to isolate
the DNA is used to cut the
plasmid
2. This results in the plasmid
having complementary sticky
ends to the sticky ends of the
DNA fragment
3. Once the complementary bases
of the 2 sticky ends are lined up,
the enzyme DNA ligase forms
phosphodiester bonds, joining
the two strands of DNA together
```
43
Why are plasmids that are used as
vectors usually given a second
marker gene?
```
It is used to show that the plasmid
contains the recombinant gene
• The marker gene is placed in the
plasmid by genetic engineering
• The plasmid is then cut by a
restriction enzyme within this
marker gene to insert the desired
gene
• If the DNA fragment is intent
successfully, the marker gene will
not function
• e.g. marker genes may be for
producing fluorescence, or an
enzyme that causes a colour
change in the presence of a
particular medium, instead of for
antibiotic resistance (which was
used in the early days)
```
44
How is the vector transferred?
The plasmid with the recombinant
DNA must be transferred into the
host cell in a process called
transformation
45
What are the methods of
| transformation?
```
Culturing the bacterial cells and
plasmids in a calcium-rich solution
and increasing the temperature
• Causes the bacterial membrane to
become permeable and then the
plasmids can enter
Electroporation
• A small electrical current is applied
to the bacteria
• This makes the membranes very
porous, and so the plasmids move
into the cells
• Can also be used to get DNA
fragments directly into eukaryotic
cells
• The new DNA will pass through
the cell membrane and the nuclear
membrane to fuse with the nuclear
membrane
```
46
What are the drawbacks of
| using electroporation?
```
• The power of the electric current
has to be carefully controlled, or
the membrane will be permanently
damages or destroyed, which
destroys the whole cell
• It is less useful in whole organisms
```
47
What is another way of
producing genetically modified
(GM) cells?
```
• Tiny electric current are applied to
the membranes of 2 different cells
• This fuses the cell an nuclear
membranes of the 2 different cells
together to form a hybrid or
polyploid cell containing DNA from
both
• This is used to make GM plants
```
48
Why is electrofusion used
| differently in animal cells?
```
• Animal cells don’t fuse as easily
and effectively as plant cells
• Their membranes have different
properties
• Polyploid animal cells (especially
mammalian ones) don’t usually
survive in the body of a living
organism
```
49
How is electrofusion used in
| animal cells?
In the production of monoclonal
| antibodies
50
Describe the varying difficult of
genetic engineering in different
organisms
```
• Much easier to carry out genetic
modification of prokaryotes than
eukaryotes
• Among eukaryotes, plants are
easier to work with than animals
```
51
How have prokaryotes been
| genetically engineered?
```
Bacteria and other
microorganisms have been
modified to produce substances
that are useful to people
• Hormones e.g. insulin and growth
forming
• Clotting factors for haemophiliacs
• Antibiotics
• Pure vaccines
• Enzymes used in industry
```
52
How have plants been
| genetically engineered?
```
Using a bacterium that causes
tumours in healthy plants
• The desired gene - e.g. pesticide
production, herbicide-resistance,
drought-resistance, or higher yield
- is placed in a plasmid of the
bacterium
• This is then carried directly into the
the plant cell DNA
• The transgenic plant cels form a
callus, which is a mass of GM
plant cells, each of which can be
grown into a new transgenic plant
```
53
How else can transgenic plant
| cells be produced?
```
• By electrofusion
• Cells made have chromosomes from
both the original cells and so are
polyploid
Stages:
1. Removal of the the plant cell wall
by celluloses
2. Electrofusion to form a new
polyploid cell
3. Use of plant hormones to
stimulate growth of a new cell wall
4. Callus formation and the
production of many cloned,
transgenic plants
```
54
How have animals been
| genetically engineered?
```
• Harder to engineer the DNA of
eukaryotic animals because animal
cell membranes are harder to
manipulate than plant cell
membranes
• The technique is used to enable
animals to produce medically
important proteins, and to try and
cure human genetic diseases e.g.
CF and Huntington’s disease
```
55
What are the uses of genetic
manipulation of
microorganisms?
```
GM microorganisms produce
substances such as insulin and
vaccines
• They are also used to store a living
record of the DNA of another
organism in DNA libraries
• Used as a tool in research for
developing new medical
treatments and industrial
processes, as well as the
development of gene technology
itself
• Genetic engineering of pathogens
could be used for the purposes of
biological warfare
```
56
What are the ethical concerns
of genetic manipulation of
microorganisms?
```
Initially some people were
uncomfortable with inserting
human genes into
microorganisms, but the pure
human medicine, antibiotics, and
enzymes produced are now seen
as overwhelmingly beneficial
• Relatively little ethical debate
about the use of GM
microorganisms except for the
manipulation of pathogens in
biological warfare
```
57
Give an example of a
| genetically modified plant
```
Insect resistance in GM soya beans
• Major world crop, and over half of
the plants are from GM strains
• Scientists have inserted a gene
into soya beans so that they
produce the Bt protein
• The Bt proteins is toxic to many of
the pest insects that attack the
plant
```
58
What are the benefits of GM
| crops?
```
• Pest-resistant GM crop varieties
reduce the amount of pesticide
spraying, protecting the
environment and helping poor
farmers
• Crop varieties resistant to
common plant diseases can be
produced, reducing crop losses/
increasing yield
• Herbicide resistance means
herbicides can be used to reduce
competing weeds and increase
yield
• The extended shelf-life of some
GM crops reduces food waste
• Crops can grow in a wide range of
conditions / survive adverse
conditions e.g. flood resistance or
drought resistance
• Nutritional value of crops can be
increased, e.g. enhanced levels of
vitamins
• Plants could be used to produce
human medicine and vaccines
```
59
What are the risks of GM
| crops?
```
• Non-pest insects and insecteating
predators might be
damaged by the toxins in GM
plants
• Insect pests may become resistant
to pesticides in GM crops
• Transferred genes might spread to
wild propoulajtion and cause
problems e.g. superweeds
• Biodiversity could be reduced if
herbicides are overused to destroy
weeds
• Extended shelf-life may reduce the
commercial value and demand for
the crop
• People may be allergic to the
different proteins made in GM
crops
```
60
What is the problem with
patenting in regard to GM
crops?
```
• People in less economically
developed countries will be
prevented form using GM crops by
patents and issues of technology
transfer
• The people who most need the
benefits of e.g. drought-resistant
crops, may be unable to afford the
GM seed
• Patenting may also make
harvesting seed from one year to
plant the next impossible
```
61
Give examples of GM animals
```
Swine fever-resistant pigs
• In the UK, a gene from wild African
pigs was inserted into the early
embryos of a European pig strain
• This gave them immunity to the
otherwise fatal African swine fever
Faster-growing salmon
• In the USA, GM Atlantic salmon
have received genes from fastergrowing
Chinook salmon
• The genes cause them to produce
growth hormones all year round
• They grow to full adult size in half
the time of conventional salmon,
making them a very efficient food
source
```
62
What is charming?
The use of genetic engineering in
animals to produce human
medicines
63
What are the 2 aspects of
| farming?
```
Creating animal models
• The addition or removal of genes
so that animals develop certain
diseases, acting as models for the
development of new therapies
• e.g. Knockout mice have genes
deleted so that they are more likely
to develop cancer
Creating human proteins
• The introduction of a human gene
coding for a medically required
protein
• Animals are sometimes used
because bacteria cannot produce
all of the complex proteins made
by eukaryotic cells
• Human gene can be introduced
into the genetic material of a
fertilised mammal, along with a
promoter sequence so the gene is
only expressed in the mammary
glands
• The fertilised transgenic female
embryo is then returned to the
mother
• A transgenic animal is born and
when it matures and gives birth, it
produces milk
• The milk will contain the desired
human protein and can be
harvested
```
64
What are the ethical issues
| with GM animals?
```
• Should animals be genetically
engineered to act as models
human disease?
• Is it right to put human genes into
animals?
• Is it acceptable to put genes from
another species into an animal
without being certain it will not
cause harm?
• Does genetically modifying
animals reduce them to
commodities ?
• Is welfare compromised during the
production of genetically
engineered animals?
```
65
What are the different types of
| gene therapy?
Somatic cell gene therapy
| • Germ line cell gene therapy
66
Describe gene therapy in
| humans
```
• Human disease e.g. CF,
haemophilia, and severe combined
immunodeficiency (SCIDS) are the
result of faulty (mutant) genes
• Scientists are researching ways of
replacing the faulty allele with a
healthy one
• They can remove the desired
alleles from healthy cells or
synthesise healthy alleles in the
laboratory
```
67
What is somatic cell gene
| therapy?
```
When the mutant allele with a
healthy allele in the affect somatic
(body) cells
• Viral vectors are often used
• Only a temporary solution for the
treated individual
• The healthy allele will be passed
on every time a cell divides by
mitosis, but somatic cells have a
limited life, and are replaced from
stem cells which will have the
faulty allele
• A treated individual will still pass
the faulty allele on to any children
they have
```
68
What is germ line cell gene
| therapy?
```
• Inserting a healthy allele into the
germ cells - usually the eggs - or
into an embryo immediately after
fertilisation (as part of IVF
treatment)
• The individual would be born
healthy with the normal allele in
place, and would pass it on to
their own offspring
• Has been successfully done with
animal embryos, but is illegal for
human embryos
```
