6.3 manipulating genomes Flashcards
define DNA sequencing
technique that allows genes to be read and sequenced
describe how DNA is cloned
the gene to be sequenced is isolated using restriction enzymes from a bacterium and placed into a plasmid then bacterium host enabling the DNA to be copied many times
What is the isolated DNA placed into with Fred Sanger’s method
a solution containing 4 bases, a primer and DNA polymerase and a modified base
How do you turn DNA into a single strand
by heating to 94-96C
Describe Fred Sanger’s method of DNA termination
to each dish of DNA along with the other components a modified base is added which is labelled with a radioactive isotope
the base is modified in such a way that once incorporated into the synthesis no more bases could be added and as the reaction progresses thousands of DNA strands of different lengths are produced which are passed through a gel by electrophoresis and smaller fragments travel longer so are sorted by length
the nucleotide at the end of each fragment is read according to its radioactive label
Describe the first DNA sequencing machine
It was based on Fred Sangers method but instead of a radioactive isotope flourescent dyes were used to label the bases which glowed when scanned with a laser and the light signature was identified by a computer
What are the diagrams produced by the first DNA sequencing machines called
autoradiograms
What is DNA incubated with during pyrosequencing
DNA polymerase
ATP sulfurylase
Luciferase
apyrase
APS
luciferin
activated nucleotide -ATP TTP CTP GTP
Describe the steps involved in pyrosequencing
A long length of DNA is cut into fragments using a nebuliser
lengths are degraded into single strands of DNA by heating to 94-96C
a sequencing primer is added to the DNA then incubated with DNA polymerase, ATP sulfurylase luciferase luciferin APS and an activated nucleotide
the activated nucleotide is incorporated into the strand of DNA and the two extra phosphates are released as pyrophosphate and in the presence of APS the enzyme ATP sulfurylase converts the phosphates to ATP and in the presence of ATP luciferase converts luciferin into oxyluciferin and the conversion generates visible light which can be detected
Describe how light is generated in pyrosequencing
the activated nucleotide is incorporated into the strand of DNA and the two extra phosphates are released as pyrophosphate and in the presence of APS the enzyme ATP sulfurylase converts the phosphates to ATP and in the presence of ATP luciferase converts luciferin into oxyluciferin and the conversion generates visible light which can be detected
How would you know how many of the same bases are in a row with pyrosequencing
the amount of light generated is proportional to the amount of ATP available and therefore indicates how many of the same type of activated nucleotide were incorporated adjacently
How are unincorporated activated bases degraded
by the enzyme apyrase
what is bioinformatics
a way to store a large amount of generated information
what are some applications of gene sequencing
genome-wide comparisons between individuals and species
prediction of polypeptide sequences
development of synthetic biology
where are sequenced genes stored
gene banks
what is meant by the phrase genes that work well will be conserved
As they have conferred a selective advantage so are likely to survive and remain in the genome for a long time or the genes will be in the genome of the species that have evolved from the original
species.
outline the useful applications of interspecific genome comparisons
to find out about evolutionary relationships and how closely particular species are related to each other
creation of phylogenic/evolutionary trees
outline the useful applications of comparing genomes of individuals
to find out about which diseases they are susceptible to
find out which medicines will/won’t give them side effects - personalised medicine
Variable sequences allow DNA profiling
What are SNP
single nucleotide polymorphisms - places on DNA where substitutions occur
What does methylation play a role in
majot tole in regulating gene expression in eukaryotic cells
Why might mapping methylation be useful
can help researchers to understand the development of certain diseases
Why might prediction of amino acid sequences in polypeptides be useful
By using knowledge of which base triplets code for which amino acids can help determine primary structure of proteins
the researchers need to know which part of the gene codes for exons and introns
what is synthetic biology
interdisciplinary science concerned with designing and building useful biological devices and systems
What is the ultimate goal of synthetic biology
build engineered biological systems provide food maintain human health and help enhance the environment
what are some examples of synthetic biology applications
nanotechnology - amyloid fibres for making biofilms for functions such as adhesion
biosensors - modified bioluminescent bacteria which glows if air polluted with petroleum pollutants
novel proteins - designed proteins that have been produced eg similar to haemoglobin but binds to oxygen and not CO
medicine
What are some problems associated with synthetic biology
raises issues of ethics and biosecurity
outline the useful applications of interspecific genome comparisons
To find out about evolutionary relationships – how closely related particular species are to each other; creation of
phylogenetic or evolutionary trees/classification.
define electrophoresis
process used to separate proteins or dna fragments of different sizes
describe the set up of electropheresis
agarose gel plate covered by a buffer solution and electrodes placed at each end of the tank so when connected to a power supply a current passes through
describe the movement of DNA fragments in electophoresis
dna has an overall negative charge due to its many phosphate groups and the fragments migrate towards the anode
describe the process of gel electrophoresis
DNA samples are digested in restriction enzymes
once agarose gel is set in the tank a buffer solution is added and comb is removed leaving wells at the end of the gel
a loading dye is added to the tubes containing the digested DNA
DNA and dye is added to wells in electrophoresis gel by pipette
electrodes are put into place and connected to a 18v battery for up to 6-8 hours
DNA fragments move through the gel at different speeds and smaller fragments travel faster
at the end of the period the buffer solution is poured away and dye is added to the gel which adheres to the DNA and stains the fragments
how do you separate proteins in electrophoresis
often carried out in the presence of a charged (SDS) which equalises surface charge on the molecules and allow proteins to separate as they move through the gel
how can electrophoresis of proteins be used
analyse the types of haemoglobin proteins for diagnosis of conditions such as sickle cell anaemia or leukaemia
define polymerase chain reaction
biomedical technology in molecular that can amplify a short length of DNA to thousands of millions of copies
define electrophoresis
process used to separate proteins or DNA fragments of different sizes
what are the potential benefits of modified microorganisms
GM microorganisms can make human insulin to treat all diabetics and HGH to treat children with pituitary dwarfism
What are the potential hazards of modified microorganisms
they could escape into the wild and transfer marker genes for antibiotic resistance to other bacteria
however GM bacteria are also modified so that they cannot synthesise an essential nutrient and cannot live outside the lab
what are the potential benefits of modified plants
tobacco plants were genetically modified to produce the toxin normally produced by a bacterium that is used as a pesticide
what are the potential hazards of modified plants
Bt is toxic to monarch butterflies,
However these butterflies do not take nectar from tobacco plants they feed on milkweed so theyre populations are still thriving
what does PCR rely on
DNA being made of two anti-parallel backbone strands
DNA having a 5’ and 3’ end
DNA grows only from the 3’ end
complimentary base pairing
In what ways does PCR differ from DNA replication
only short sequences can be replicated and not whole chromosomes
requires addition of DNA primer not RNA
cycle of heating and cooling is needed to separate the DNA strand, bind primers and for DNA strands to be replicated
why was the initial PCR method very time consuming
As DNA was heated to denature the histone proteins and then allowed to cool to 35C to anneal the primer and allow DNA polymerase to work
Describe the method of PCR
sample of DNA is mixed with DNA nucleotides, primers, magnesium and Taq DNA polymerase
mixture is heated to 94-96C to break the hydrogen bonds and denature double strands into single strands
Taq DNA polymerase can now bind to the primer and temperature is raised to and kept at 72C as that is the optimum for Taq DNA polymerase allowing it to add nucleotide strands in the 5’3’ direction generating a new strand
1-2-4-8-16 etc
What are the applications of PCR
tissue typing
detection of oncogenes’
identifying viral infections
monitoring spread of disease
forensic science
research
How does the Taq DNA polymerase differ from the DNA polymerase in regular replication
it has a higher optimum temperature of 72C
What is the procedure of DNA profiling
DNA is obtained from the sample and PCR must be done to amplify the number of copies
DNA is then digested with restriction enzymes which cut either side of the STR and the fragment size will vary between individuals
fragments are separated by electrophoresis and a banding pattern can be seen
DNA of which the individual is being compared to goes through the same steps and banding patterns are compared
What would the banding patterns of two related individuals look like
they would have some bands in the same area and wouldn’t have some
What are some applications of DNA profiling
forensic science
maternity and paternity disputes
analysis of disease
What are short tandem repeats
highly variable short lengths of repeating DNA and the number of STR varies from person to person and 13 are analysed
What is a DNA probe
a short single stranded length of DNA that is complimentary to a section of DNA being investigated
What may a DNA probe be labelled with
a radioactive marker such as P32 that can be revealed by exposure to photographic film
Fluorescent marker that emits a colour on exposure to UV light
How are probes useful in locating specific DNA sequences
locate a specific gene to be used in genetic modification
to identify same gene in a variety of genomes
to identify presence or absence of specific allele
What are microarrays
a number of probes are put on a fixed surface known as a microarray and by applying the DNA under investigation the surface can reveal the presence of multiple alleles that match the fixed probes because the sample DNA will anneal to the complementary fixed probes
Why is a reference used in microarrays
to ensure that the mechanism is working
What must the DNA sample undergo before it goes on a microarray
must be broken into smaller fragments and undergo PCR
what is a potential benefit of modified soya beans
GM soya beans resistant to a herbicide were produced so that weeds competing with the soya plants could be killed with the herbicide
What is a potential hazard of modified soya beans
potential for the gene of herbicide resistance to be passed on to the weeds however this hasn’t happened yet
What is a potential benefit of modified golden rice
golden rice was modified to contain a gene from daffodils so beta carotene would be present in rice preventing deaths from lack of beta carotene in children
What is a potential hazard of modified golden rice
concern that farmers would have to buy the seed every year, but the company has offered free licenses to farmers
What is a potential benefit of modified plantains
Plantains are being nutritionally enhanced with zinc so that areas where there is little meat consumption do not become deficient as zinc is a cofactor and essential for regulating insulin secretion
What is a potential hazard of modified plantains
some people fear eating food that contains foreign DNA and worry the inserted genes will somehow be expressed in us
What is a potential benefit of crop plants resistant to pests
so that farmers don’t need to use pesticides when they sow seeds making it better for the environment and safer for the farmers as they get less exposure to the pesticides
What is a potential hazard of crop plants resistant to pests
there were concerns that local farmers would not want to buy GM seed
What is a potential benefit of modified pathogens
viruses gm’ed to have no virulence can be used to make vaccines
can also be used as vectors in gene therapy
What is a potential hazard of modified pathogens
there have been many problems with the use of viruses in gene therapy as the allele may be inserted into the genome in a way that increases the risk of cancer or interferes with gene regulation
What is a potential benefit of modified mice
millions of GM mice have been bred for medical research and used to develop therapies for breast and prostate cancer
Others have genes knocked out to see theyre functions
What is a potential hazard of modified mice
some people object to use of animals for medical and pharmaceutical testing
What is a potential benefit of modified pharmaceutical proteins
genes for human pharmaceutical proteins can be inserted into goats or sheep and the human protein they express into the milk is collected
protein is too large for a bacterial cell to synthesise
e.g. alpha antitrypsin to treat hereditary emphisema
What is a potential hazard of modified pharmaceutical proteins
concerns for welfare of GM sheep and goats
What is a potential benefit of modified silk
genes for spider silk have been inserted into goats and produce spider silk protein in their milk and the silk can be used for cables and sutures
What is a potential hazard of modified silk
concerns about the welfare of the GM goats
define genetic engineering
combining DNA from different organisms
what does genetic engineering also define
recombinant DNA
What are the stages of genetic engineering
obtaining the gene
copy of gene is placed inside a vector
vector carries gene into recipient cell
recipient expresses the novel gene
what are the different ways that a gene may be obtained
using reverse transcriptase to catalyse formation of cDNA from mRNA and addition of primers and DNA polymerase to make double stranded DNA
if scientists know the nucleotide sequence then the gene can be synthesised using an automated polynucleotide synthesiser and PCR to amplify the gene .
DNA probe can be used to locate a gene within a genome and can then be cut using restriction enzymes
what can be used as a vector to place a gene into
plasmids
attenuated virus
describe how you would place a gene into a plasmid
plasmids can be obtained from organisms such as bacteria and mixed with restriction enzymes that will cut the plasmid at specific recognition sites leaving exposed unpaired nucleotide bases called sticky ends ‘‘if the free nucleotide bases complimentary to the sticky ends of the plasmid are added to the ends of the gene to be inserted then the gene and enzymes should anneal catalysed by DNA ligase
what are the 6 ways to get the vector in to the recipient cell
heat shock treatment
electroporation
electrofusion
transfection
T1 recombination plasmids
gene gun
Describe how heat shock treatment gets the vector into recipient cell
0C and 42C alternating temperatures in the presence of calcium chloride will cause bacteria cell walls and membranes to become more permeable because the positive calcium ions surround the negative parts of both the DNA molecules and phospholipids in cell membrane reducing repulsion between foreign DNA and host cell membranes
describe electroporation
high voltage pulse applied to cell to disrupt the cell membrane
describe electrofusion
electrical fields help to introduce DNA into cells
describe transfection
DNA can be packaged into a bacteriophage which can transfect the host cell
describe T1 recombinant plasmids
they are inserted into bacterium which infects some plant and naturally inserts its genome into the host cell genome
Describe gene guns
small pieces of gold or tungsten are coated with DNA and shot into plant cells
describe reverse transcriptase
catalyses the production of complimentary DNA using RNA as a template
describe how insulin is produced from GM bacteria
mRNA obtained from beta cells in islets of Langerhans
adding reverse transcriptase makes a single strand of cDNA with DNA polymerase makes a double strand
addition of free unpaired nucleotides at the ends of DNA to make sticky ends
with the help of ligase enzymes the insulin gene can now be inserted into plasmids extracted from e.coli which now become recombinant plasmids
e.coli bacteria are mixed with recombinant plasmids and are subjected to heat shock in the presence of CaCl2 ions so they take up the plasmids
Describe the non-recombinant plasmid in insulin production compared to the recombinant plasmid
nr contains an ampicillin-resistance gene and tetracycline-resistance gene but in a recombinant plasmid the tetracycline-resistance gene is interrupted as the human insulin gene is inserted
How would you know if the bacteria took in the plasmid
grow in ampicillin and if it doesn’t die means that plasmid is there due to presence of ampicillin resistance gene
How would you know if the insulin gene was in plasmid
grow in tetracycline if they die insulin gene is present because it is inserted in the resistance gene
what are the two types of gene therapy
somatic cell
germ line
define somatic cell gene therapy
gene therapy by inserting functional alleles into body cells
define germ line gene therapy
gene therapy by inserting functional alleles into gametes or zygotes
what is the principle of gene therapy
is to insert a functional allele of a particular gene into cells that contain only mutated or non-functioning alleles of the gene
if the inserted allele is expressed then the individual will produce a functioning protein and no longer have symptoms associated with the generic disorder
what is the difference between somatic cell and germ line therapy
somatic cell alterations in the genome are not passed onto the offspring
what can be used to enter the functional alleles to the non-functional allele
liposomes
viruses
artificial chromosomes
describe liposomes
small spheres of lipid bilayer
how are liposomes used to treat cystic fibrosis
they are placed in aerosol inhaler and sprayed into the noses of patients some will pass through the plasma membrane of cells lining the respiratory tract
if they also pass through the nuclear envelope and insert into the host genome and the host cell will express the CFTR protein - a transmembrane chloride ion channel
how can viruses be used as vectors
a virus is genetically modified so that it encases the functional allele to be inserted into the patient whilst at the same time being unable to cause the disease it can enter the recipient cells taking the allele with it
what are the potential problems of using viruses as gene delivery agents
may still provoke an immune or inflammatory response on the patient
the patient may become immune to the virus making further deliveries difficult
increased risk of cancer by inserting allele in a location that disrupts gene involved involved in regulating cell division
how can artificial chromosomes be used in gene therapy
possibility of inserting genes into an artificial chromosome that would co-exist with other chromosomes in the target cell
describe the action of germ line therapy
involves altering genome of gametes or zygotes altering all the cells of the individual and offspring may also inherit the foreign allele so it has potential to change genetic makeup of many people
what are the concerns of germ line gene therapy
about how the genes are inserted - they may find their way to a location that could disrupt the regulation and expression of genes
ethically impermissable
