Unit 19: Gene Tech (JW) Flashcards
define the term recombinant DNA
DNA made by combining 2 or more DNA pieces, each from a different organism/source
explain that genetic engineering is the deliberate manipulation of genetic material to modify specific characteristics of an organism and that this may involve transferring a gene into an organism so that the gene is expressed
Deliberate manipulation of genetic material of a living organism
to modify specific characteristics of an organism
involves transfer of a gene into an organism so that the gene is expressed in the recipient organism
e.g. human insulin gene -> bacteria
Outline the general process of geneting engineering
1 manipulate DNA to modify organism’s characteristics / AW ;
2 gene / allele / (section of) DNA, obtained using restriction, enzyme / endonuclease ;
3 use of reverse transcriptase to make, gene / allele / (section of) DNA, using mRNA ;
4 gene / allele / (section of) DNA, inserted into, vector / plasmid, using ligase ;
5 add / insert, (recombinant), vector / plasmid, into, (host) cell / bacterium ;
6 clone / multiply, cell / bacteria ; A put bacteria in a fermenter
7 gene is expressed and, protein / factor XIII / factor IX, is made ;
8 AVP ; e.g. artificial / chemical, synthesis of new gene
Outline how genetic engineering gave maize the trait of insect resistance
gene from another species
restriction enzyme
cuts plasmid
DNA ligase
forms recombinant plasmid/vector/DNA
new gene enters maize’s DNA/cells/genome
gene expressed/transcribed
to produce protein
marker genes / insert promoter
3 sources of genes to be transferred into an organism
• extracted from the DNA of a donor organism
• synthesised from the mRNA of a donor organism
• synthesised chemically from nucleotides
What is the advantage of synthesising gene from mRNA of organism?
- easier to find mRNA than finding gene as specialised cells produce very specific type of mRNA
- easier to extract in cytoplasm compared to DNA in nucleus
- mRNA does not contain introns (non-coding regions of DNA)
Explain the role of restriction endonuclease in genetic engineering
cut DNA
at specific restriction site
palindrome
sticky/blunt ends
Explain the role of DNA ligase in genetic engineering
joins desired gene and plasmid together
seal sugar-phosphate backbone
forms phosphodiester bonds
make recombinant DNA
Outline structure of plasmid
Small, circular double-stranded DNA
Explain why plasmids are frequently used in gene technology
small circular piece of double stranded DNA
replicate independently
high copy number
easy to extract from bacteria
can be cut using restriction enzyme
gene inserted
taken up by bacteria
may contain marker genes
identify transformed bacteria
acts as vector
may carry promoter
can be artificially produced
Identify and explain properties of plasmids that allow them to be used as vectors
small so can enter cells
self-replicate so multiply
have restriction sites - so can be cut by restriction enzymes
have marker genes so transformed cells can be recognised
have promoter so gene can be expressed/transcribed
circular so more stable - not damaged by host cell enzymes
Explain the role of plasmids in genetic engineering
plasmid has complementary sticky ends to sticky ends on desired gene fragment
as it was cut by same restriction enzyme used to cut desired gene
joined by CBP and H bonds forming
Describe the process of transformation
plasmids transferred to host cells
Bathing plasmids & bacteria in ice-cold calcium chloride solution
briefly incubate at 40ºC
makes bacteria membrane permeable
Electroporation - bacteria given small electric shock - make membranes very porous
Explain the role of DNA polymerase in genetic engineering
uses ss cDNA to produce ds DNA
DNA polymerase replicates DNA
Explain the role of reverse transcriptase in genetic engineering
make ss cDNA from mRNA
produced by retroviruses
Describe role of promoter in gene expression
RNA polymerase binds to promoter
TF binds to promoter
increases transcription
correct strand/template is transcribed
Explain why a promoter is included in the genetic package
So inserted genes are expressed
RNA polymerase binds at promoter
mRNA synthesis / controls gene expression
correct template / strand
control quantity of protein produced
control where/which part of plant makes protein
Explain why differences in the control of gene expression in prokaryotes and eukaryotes mean that expression vector plasmids must contain a prokaryotic promoter
eukaryote & prokaryote promoter sequences are different
eukaryote & prokaryote RNA polymerase enzymes are different
prokaryotic RNA polymerase only binds to prokaryotic promoter / does not recognise eukaryotic promoter
so no transcription
eukaryotic promoter requires binding of TF that are not present in prokaryotes
explain how gene expression may be confirmed by the use of marker genes coding for fluorescent products
Marker gene
identify which bacteria have successfully taken up plasmid / been transformed
fluoresces under UV light
explain what is meant by gene editing
type of genetic engineering
involve insertion, deletion or replacement of DNA at specific sites in the genome
Diff btwn genetic engineering and gene editing
gene editing involves modification of existing DNA, whereas genetic engineering involves insertion of DNA from another organism
Explain the social benefits of this example of gene editing
increase yield
improves quality of crop
helps solve global demand for food
more income for farmers
cheaper
less cost of pesticides
describe and explain the steps involved in the polymerase chain reaction (PCR) to clone and amplify DNA
rapid amplification of DNA
only small amt of DNA required
denaturation at 95ºC
breaking H bonds, splitting DNA into 2 ss DNA template strands
annealing at 60-65ºC
primer added
binding to bases on template strands by CBP
extension at 70-75ºC
Taq polymerase builds new strands
by adding free nucleotides
Taq polymerase is heat stable
does not need replacing each cycle
strand replicated - 2 new DNA strands produced
new strands denatured - process repeats
efficient process
Explain the role of Taq polymerase in PCR
Taq polymerase builds new strands
by adding free nucleotides
Taq polymerase is heat stable
does not need replacing each cycle
Explain why primers are used in PCR
primers bind to specific bases on DNA by CBP during annealing
so that so that Taq polymerase can bind to DNA -> replicate DNA
Explain how gel electrophoresis separates DNA fragments cut with restriction endonucleases.
DNA phosphate group -vely charged
moves to anode
when current applied
larger fragment move more slowly
due to resistance
buffer solution
describe and explain how gel electrophoresis is used to separate DNA fragments of different lengths
DNA cut with restriction enzymes
DNA fragments placed in well in gel AT CATHODE
current applied
fragments negatively charged due to phosphate group
move towards positive electrode/anode
gel acts as a molecular sieve
smaller fragments move faster than larger ones
current switched off
Southern blotting
Staining for visualisation
compare banding patterns to reference bands
alleles have diff positions on gel
outline how microarrays are used in the analysis of genomes
obtain ssDNA
label ssDNA with fluorescent dye
probes on microarray
probes are short lengths of ssDNA
complementary to DNA being tested for
many copies of 1 type of probe placed in each cell of microarray
DNA binds to/hybridises with probes
unbound/excess DNA washed off
UV light used to detect presence of fluorescence
fluorescence indicates presence of gene
intensity of fluorescence indicates level of gene expression
positions recorded by laser/scanner
positions identified as named genes
outline how microarrays are used in detecting mRNA in studies of gene expression
probes are ss DNA
each probe unique to a particular gene
probes correspond to thousands of different genes
extract mRNA from 2 samples
mRNA used as template to make cDNA using reverse transcriptase
cDNA linked to fluorescent dye
cDNA added to microarray
cDNA binds to probes
by complementary base pairing
excess cDNA washed off
exposed to UV light
fluorescent shows expressed genes
intensity of fluorescence shows level of gene expression
positions recorded by laser/scanner
positions identified as named genes
outline the benefits of using databases that provide information about nucleotide sequences of genes and genomes, and amino acid sequences of proteins and protein structures
bioinformatics is a large database of DNA sequence data
for comparison
to search for DNA sequence similar to human protein
modelling/predicting 3D protein structure
DNA sequence data from all around the world
fast, efficient, accurate
determine percentage similarity
Using recombinant insulin to treat diabetes
bacteria plasmids modified to include human insulin gene
recombinant plasmids inserted into E coli by transformation
once transgenic bacteria are identified, they are isolated, purified and placed into fermenters that provide optimal conditions
transgenic bacteria multiply by binary fission
transgenic bacteria express the gene coding for insulin
insulin extracted and purified
Using recombinant factor VIII to treat haemophilia
kidney & ovary hamster cells genetically modified to produce factor VIII
once modified, recombinant cells placed into fermenter and cultured
optimal conditions in fermenter -> hamster cells constantly express factor VIII protein
factor VIII protein extracted and purified
injectable treatment for haemophilia
Using recombinant adenosine deaminase (ADA) to treat Adenosine Deaminase Deficiency
larva of the cabbage looper moth has been genetically modified (using a virus vector)
produce ADA enzyme
used as treatment whilst patients wait for gene therapy / if gene therapy not possible
explain the advantages of using recombinant human proteins to treat disease
identical to human protein
produce in larger quantities
no allergic reaction / side effects
human protein may have higher activity
no chance of developing tolerance
no risk of transmitting diseases
unlimited supply
lower cost of production
quicker to produce
no ethical/religious issues
easier to obtain purified product
outline the advantages of genetic screening, using the examples of breast cancer (BRCA1 and BRCA2)
early treatment
lifestyle changes
elective mastectomy
regular checkups
prevents unnecessary prolonged suffering if discovered and treated early
prevents early death
removes worry
can choose whether to have children
preventative = cheaper than later treatment
outline the disadvantages of genetic screening of breast cancer (BRCA1 and BRCA2)
may cause worry
person may not develop cancer
test is expensive - not available for everyone
social/financial discrimination - implications for life insurance
may decide not to have children
Other advantages of genetic screening of diseases
quickly start treatment after birth
informed decision made about continuing with pregnancy (if screened before birth)
mentally prepare for cost of treatment
does not rely on family knowing family history of SCID
(example of SCID) prevents child with SCID from developing infections before diagnosis
Explain why Huntington’s disease cannot be treated with gene therapy
dominant allele
so will still be expressed even in presence of recessive allele
gene therapy only used to treat recessive diseases
cannot remove dominant allele
dominant allele affects tissues in many part of the body
Explain what is meant by gene therapy
treat diseases such as SCID, CF
caused by faulty/recessive allele
deliver gene into target cell of individuals
Suggest the main steps involved in creating recombinant DNA for gene therapy
synthesise dominant allele from mRNA using reverse transcriptase + DNA polymerase
probe / electrophoresis for identification
PCR to amplify DNA
restriction enzyme
DNA ligase - to join desire gene + plasmid
add promoter
Explain why the fact that LCA is an autosomal recessive genetic disease makes it suitable for treatment with gene therapy
add / insert correct dominant allele
only need 1 allele
to cure disease
synthesise correct functional protein
no need to remove faulty allele
Suggest how children with ADA-deficient SCID can be treated with gene therapy using a virus
obtain normal, dominant allele
insert allele into AAV virus as vector
remove stem cells / T-lymphocytes
insert allele into stem cells / T-lymphocytes
return cells to body by injection
Outline the challenges of using a virus for gene therapy
can insert healthy allele randomly into host DNA
may cause cancer
inserted allele may be inactivated
virus may not enter target cells
discuss the challenges in using naked DNA as vector for gene therapy
- must be injected into target cell
- low efficiency of cellular uptake
- rapidly broken down
discuss the challenges in using viruses as vector for gene therapy
- small packaging capacity - only small amt of DNA carried
- low probability of integration into host genome
- cause mutations in host DNA
discuss the challenges in using liposomes as vector for gene therapy
- low ability to add DNA into target cells
Outline social or ethical implications of screening embryos by embryo biopsy
embryos may be destroyed
wrong for parents to choose - designer babies
contrary to beliefs/values
less chance of HTT being passed on
people with faulty allele who otherwise would not have children can now do so
discuss the social and ethical considerations of using gene therapy in medicine
potential side effects that could cause death
germline gene therapy - causes modification to be passed on to future generations
commercial viability for pharmaceutical companies: if it is a rare disease, the relative small number of patients may not mean that the companies will make a profit
expense of treatments as multiple injections of the genes may be required if the somatic cells are short-lived
possibility that people will become less accepting of disabilities as they become less common
Who has the right to determine which genes can be altered and which cannot
discuss the social and ethical considerations of using genetic screening in medicine
taking preventative measures - elective mastectomy
Using pre-implantation genetic diagnosis to select embryos that do not carry faulty disease - designer babies
risk of miscarriage due to procedures used to collect DNA
Choosing to terminate a pregnancy because the embryo has a genetic disorder
make informed reproductive decisions
possibility of stigmatisation and discrimination
Suggest what steps will be needed to make identical genetically modified AD organisms
obtain normal allele from mRNA
by producing ss cDNA from mRNA using reverse transcriptase
DNA polymerase to produce ds DNA
restriction enzyme
use vector
on zygote
add promoter
add marker gene
cloning/embryo splitting
Outline how genetic engineering produces GM salmon
gene for growth hormone inserted
promoter from another species also inserted
growth hormone produced all year round
GM salmon grow faster and larger
increase salmon yield
growth hormone gene + promoter gene
promoter gene ensured growth hormone continually being expressed
To prevent the GM salmon from reproducing in the wild, all the salmon are female and sterile
Outline how genetic engineering produces herbicide-resistant soybean plants
allows farmers to spray a herbicide on the crop after germination to kill weeds
that would otherwise compete with the growing soybeans for light, water and minerals
resistant gene comes from Agrobacterium
gene allows an enzyme in the soybean to continue to synthesise 3 AA needed to produce proteins required in the growing tips of plants
herbicide glyphosate inhibits the enzyme in plants without the resistant gene; without the proteins being synthesised, the plants die
Outline advantages of growing insecticide/herbicide resistant plants/organisms
insect is killed by the herbicide/insecticide
reduces competition
manually spraying insecticide/herbicide is difficult + expensive
Outline how genetic engineering produces Bt cotton plants
Bt toxin gene obtained from Bacillus thuringiensis
restriction enzyme
amplify gene using PCR
insert gene into plasmid
add promoter
seal plasmid using DNA ligase
forms recombinant DNA
cotton cell takes up plasmid
expresses new gene - Bt toxin gene transcribed
Advantage of GM Bt cotton plants
Bt toxin only kills specific insects
reduce use of insecticides - less costs to farmers
Disadvantage of GM Bt cotton plants
Insect populations have developed resistance to the genes for Bt toxin, reducing its effectiveness
resistance may be transferred to wild plants
contamination of food marketed as organic
may kill pollinators/beneficial insects
decrease in biodiversity
potential health risks for humans
Problems with producing medicinal drugs from the milk of genetically modified goats
possible side effects/allergy - may not be pure
process may harm goats - animal welfare
discuss the ethical and social implications of using genetically modified organisms (GMOs) in food production
GM production cost is expensive - only 1% success
monopoly held by MNCs
people may avoid/refuse to buy GM food
no long-term studies done on effects on human health - unknown health consequences
possible allergic reactions/immune response/side effects
more food supply
GM crops decrease food cost
increase country’s wealth
relieve hunger / starvation
reduce land area for crops - protect biodiversity
less insecticides used
less money spent on insecticides
high cost of GM seed
decrease in genetic variation
Discuss the social implications of using genetically modified organisms in food production
increase yield
increased quality
improvement to health
longer shelf-life
some GM crops are adapted to unfavourable conditions
less cost on insecticides
consumer resistance
possible allergen/side effects
expensive
may have to buy seeds every season
contaminate food labelled as organic
