Genetic technology Flashcards
What is Polymerase chain reaction (PCR) for and what are the components used?
*Rapid and efficient process
*Function:clone and amplify DNA
*Amplify=production of many copies of a length of DNA
*Only small sample of DNA needed
Components needed;
1. Template DNA
2. Taq polymerase
*Hest-stable DNA pol from thermus aquaticas bacteria
3. Buffer
*Contains KCl and MgCl2
4. 4 types of nucleotides (A,T,C,G)
5. 2 primers
*Short sequence of -20bp of single stranded RNA/DNA
*Complementary base pair to start and end end of target region for amplification
How does Polymerisation chain reaction work?
- Denaturation (95°C)
*DNA strands separate/denature into 2 strands by heat
*Hydrogen bonds between DNA strands breaks
*Bases are exposed
*Produce template strands for copying - Annealing (60-65°C)
*Primer added
*Primers anneal/bind to specific section of DNA
*Via complemetary base pairing
*New hydrogen bonds form
Role of primers:
*Bind to target region for amplification
*Acts as a staring point for Taq polymerase to bind
→Taq polymerase only binds to double-stranded DNA and add new nucleotides to an existing strand
*Reduce reannealing of separated strands
- Extension (70-75°C)
*Taq polymerase binds to primer
*Synthesises new DNA strands
*Complementary to the DNA template strands
*Taq polymerisation has a high optimal temperature/is heat stable
*Does not need replacing each cycle
*DNA is heated again to separate strands
*The process is repeated until sufficient DNA is produced (Usually 30 cycles)
*The number of DNA molecules doubles every cycle=efficient process!
*Number of DNA double helix copies made from one starting molecule after n cycles of PCR = 2^n
What are the advantages and disadvantages of Polymerase Chain Reaction (PCR)?
Advantages:
*Rapid, effecient
*Only small sample of DNA needed for amplification of DNA
*Process is automated in a thermal cycle
Disadvantages:
*Need to know the precise DNA sequence beforehand to design primers
*DNA amplification have to be in shorter fragments than gene cloning in bacteria (genetic technique 2)
What are the applications of the polymerase chain reaction?
Function: clone and amplify DNA
Applications:
*DNA sequencing
→Able to amplify small amount of DNA extracted for sequencinh (e.g. in fossils, blood sample)
*DNA profiling
→Able tp amplify small amounts of DNA extracted (E.g. at crime scene)
*Recombinant DNA technology
→Amplify DNA/gene needed for insertion into plasmid
*Genetic screening
→To identify mutations/disease genes/DNA from pathogens
→Use primers complementary to targer gene
→To identify and amplify target gene in specific
→Gel electrophoresis used to isolate gene
What are the steps of recombinant DNA technology?
- Identify and isolate mRNA/DNA from organism
- Cut target gene and plasmid DNA
- Recombine/join gene to plasmid DNA
- Insert recombinant plasmid into bacteria
- Identify modified/transformed bacteria
- Grow bacteria in fermenters for large-scale production.
Describe the steps of recombinant DNA technology in the production of human insulin.
- Obtain mRNA for human insulin
*From beta cells of islets of langerhans of panreas
*Reverse transcriptase to make cDNA
(single stranded complementary DNA) from mRNA)
*DNA polymerase used to make double stranded cDNA from single stranded cDNA
*Final result:gene with no introns (non-coding regions), shorter DNA
DNA may be amplified using polymerase chain reaction(PCR - Cut target gene and plasmid DNA
*use restriction enzyme to cut the gene
*restriction sites should be present at both sides of gene
*Obtain plasmids from bacteria
*Cut plasmid at 1 restriction site using SAME restriction enzyme
*Complementary sticky ends produced - Recombine/join gene to plasmid DNA
*Mix cDNA/insulin gene with plasmid
*DNA ligase seals nicks in sugar-phosphate backbone
→Catalyses the formation of phosphodiester bond
*To form recombinant DNA - Insert recombinant plasmid into bacteria
1)Recombinant plasmids mix with bacteria
2)Treat bacteria with a solution of Ca2+ ions and allow to cool
3)Apply heat shock (42°C) or use electroporation to increase chances of plasmids passing through cell surface membrane
Only 1% bacteria will take up recombinant plasmids=transformed
5. Identify modified/transformed bacteria
*Marker genes in the plasmids helps to recognise recombinant plasmids/modified bacteri/transgenic organisms
*e.g. genes of interest is inserted close to OR into marker gene
*Both target gene and marker gene are expressed
If gene of interest is inserted into the marker gene, then the marker gene is distrubted. There is another marker gene that is expressed together. Extra function: easy to identify recombinant plasmid
Ways things can go wrong:
1. bacteria did not take up recombinant plasmid
2. Gene of interest did not jpoin with plasmid…cut plasmid just rejoined with itself
- Grow bacteria in fermenters for large-scale production
*Allow transformed bacteria cells to multiply/clone
*Grow im large-scale culture/fermenter
*E.coli can divide once every 20 mins
*bacteria produces multiple copies of the gene/protein product (in this case, human insulin)
*bacteria has replication, transcription and translation machinery to copy the gene and express the protein product (e.g. DNA polymerase, RNA polymerase, ribosomes)
*Insulin extracted and purified to be sold on market
What is a reaction enzymes/endonucleases?
*From bacteria
*recognize, bind and cut DNA at a specific sequence, called a restriction site
*Hydrolyses/leaves the phosphodiester bond btwn nucleotides
*Diff kind of RE cuts at a diff specific seq
*Most restriction sites are palindromic sequences (this sequence reads the same in both directions)
*Restriction enzymes produce sticky ends or blunt ends
At the “sticky ends”:
*result of a staggered cut
*A few unpaired nucleotides at ends
*Able to easily form H bonds/complementary base pairs
At “blunt ends”:
*No unpaired nucleotides
What are plasmids?
*Small circular, double stranded DNA
*Found in bacteria, but now usually artificially made in lab
Frequently used in gene technology because:
*Small so can be inserted into cells
*Circular so more stable/not damaged by host cell enzymes
*Plasmids act as vector to deliver desired genes to bacteria
*Easy to extract from bacteria
*Can be taken up by bacteria due to low molecular mass/small
*Has high copy number-many copies can be present in one bacterial host cell
*Replicate independently within bacteria-able to clone/replicate any genes inserted into them
*Has specific DNA sequences needed!
What are the specific DNA sequnces we need, that are found in plasmids?
Origin of replication
Restriction site
Promoter
Marker gene
What is the function of origin of replication DNA sequence in plasmids?
DNA sequence:Origin of replication
Function:Allows bacterial DNA polymerase to bind an replication to be initiated
What is the function of restriction site DNA sequence found in plasmids?
DNA sequence: Restriction site
Function:
*For restriction enzyme to cut and produce sticky ends, so gene of interest can be inserted
*Can have multiple to be cut by different restriction enzymes→has multiple cloning sites
What is the function of promoter DNA sequence found in plasmids?
DNA sequence: Promoter
Function:
*Initiates transcription
*Allows binding of RNA polymerase/transcription factors
*Ensures correct strnd is used as template
*Diff promoters determine diff level of expression and where it is expressed
*E.g. use promoter upstream of lacZ gene in lac operon-target gene will be expressed in the presense of lactose
What is the function of the marker gene DNA sequnce dound in plasmids?
DNA sequence: Marker gene
Function:
*gene of interest is inserted close to/into marker gene
*Both the target gene and marker gene are expressed
*Helps to recognise recombinant plasmids/modified bacteria/ transgenic organisms
*E.g. genes for antibiotic resistance, genes for fluorescent/easily stained substances
What are the ways to identify transformed bacteria?
a)Use antibiotic selection
b)Use green fluorescent protein (GFP)
c)Use an easily stained substance (beta galactosidase, GUS)
How is antibiotic selection used to identify transformed bacteria?
*genes for antibiotic resistance used as markers
e.g. ampicillin resistance gene (amp^r), tetracycline resistance gene (tet^r)
*Grow on agar containing the antibiotic (ampicliin)
*Bacteria with plasmid will be able to survive
*Bacteria without plasmid dies
*Then make a replica plate by using a sponge/velvet pad
*Grow bacteria on agar containing 2nd antibiotic (tetracycline)
*Bacteria with recombinant plasmid will die
BUT PROBLEM!
*Risk of spread of antibiotic resistance to other bacteria of same/diff species
*Plasmids are easily transferred between bacteria via conjugation
*This makes the use of antibiotics less effective in disease-causing bacteria
*Slower process for identification of transformed bacteria as well
*So… use other methods to identify modified bacteria
How can we use green fluorescent protein (GFP) to identify transformed bacteria?
*Gene for fluoroscent substances used as marker e.g. gene for green fluoroscent protein (GFP)
*From jellyfish
*GFP emits bright green light
*When exposed to UV
*Bacteria with plasmid will express GFP and will appear green
How can we use an easily stained substance to identify transformed bacteria?
*Use gene that codes for asily stained subtances as marker
e.g. gene for beta galactosidase aka lacZ gene
e.g. gene for GUS enzyme
*Enzyme splits with a special non-blue substrate into product that is blue
*Bacteria with plasmid will become blue
What are the advantages of using genes for fluoroscent/easily stained substances as markers
*Avoid use of antibiotics
*More economical/time saving/labour saving
*Visible colour is easy to identify/detect
*Enable identification of transformed cells AND transgenic organisms
*No known risk/ill effect on GM organism
What are the applications of recombinant DNA technology?
*Conventional method=extract insulin from pancreas of pigs or cattle
Advantages of producing human insulin by gene technology:
1. Chemically identical to human insulin
→exact fit to insulin receptors on target organs
→Does not stimulate the immune system
→Faster response
→Fewer side effects
→Less/no risk of allergic reaction
2. Effective in people who developed tolerance to animal-derived insulin
3. Avoid ethical issues related to religion and use of animal products →no killing of animals
4. Lower cost of purification and processing
5. Mass production=large and constant reliable supply all year around
6. Less risk of contamination/infection
→no risk of transfer of disease
7. Potential to engineer/improve recombinant proteins
What are the applications of recombinant DNA technology?
*Production of pharmaceuticals
→No modifying of protein in bacteria (bcs no membrane-bound organelles)
→Can genetically modify eukaryotic cells to produce human proteins
E.g. yeast cells, bacteria, insect larvae cells, mammalian cells
Human protein:Human insulin, Treatment for: diabetes, produced in: recombinant bacteria
Human protein: factor VII, treatment for: helps blood clot in haemophillia patients, produced in: GM hamster kidney and ovary cells (in fermenter)
Human protein: Adenosine deaminase (ADA), treatment for: help in development of B and T cells in severe combined immunodeficiency disorder (SCID) patients, produced in: GM insect larva of the cabbage looper moth
*used in genetic engineering to produce GMO’s in agriculture
What is genetic engineering?
*Involves manipulation of naturally occuring processes and enzymes
*Extraction of genes from one organism/synthesis of genes
*In order to place them in another organism
*Of the same or diff species
New Hosts:
*Have recombinant DNA (rDNA)=combination of DNA for two or more sources
*Expresses the new gene product/protein
*Genetically modified (GM)/transgenic organisms =organisms with any altered DNA
Describe genetically modified organisms in agriculture.
*Aims of using genetic engineering to produce GMO’s in agriculture:
*Improve quality of crop plants and livestock
*Increase yield of crop plants and livestock
→Solve world food demand
Livestock can be engineered to:
*Have high growth rate
*Grow larger
*Have highe yeild (milk, meat)
Crop plants can be engineered to:
*Have higher yeild
*Better quality/taste
*Delayed ripening of fruits (to increase shelf life)
*Additional, nutritional benefits
*Resist disease/pests/insects (so less pesticides used)
*Resist herbicides (to reduce competition from weeds)
*Grow in adverse conditions/more tolerant to climate change (e.g. hot, cold climates)
*Grow in poor quality land, require less fertilizer
→More cost effective/have health benefits
→Less effect on food chain/pollinators
Why do we use GMO’s instead of artificial selection?
*Much faster results
*Allows the retention of other desirable characteristic of the best varieties of species
*Able to use the best genes from other species of plants and even non-plants
BUT….
*Complicated process
*Expensive
*Not always successful
Describe genetically modifed atlantic salmon
*genes for growth hormone regulator transferred from diff species of salmon
*Genes for promoter from another diff species
Benefits:
*High yeild
*Consistent yeild all-year around
*Conserve wild fish populations
*All modifies salmon egss are triplod and sterile
→So impossible for them to breed amongts themselves and with other salmon
→Eliminate impact to wild population
Describe insect resistant crops-Bt crops
E.g. Bt cotton, Bt maize
*Bt cotton protected against boil weevils
*Bt maize protected against corn borer caterpillars
*Corn borer eats leaves and burrows into stalk
→Plant cannot support the ears of corn
*Gene for Bt toxin from bacteria bacillus thuringiensis inserted into maize/cotton
*Lethal to insects but not other animals
*Crops able to produce own insecticide
What are the benfits of Bt crops?
*Increased yeilds
*Only kills specific insects that eats it and does kill beneficial insects
E.g. pollinators/bees/predators of pests
→Conserve biodiversity/food web
*Less pesticide used
→Reduce risk of pesticide affecting non-target species in the same enviroment
*Less risk of harm from spray drift/pesticide residues on food
Describe herbicide-resistant crops-glyphosphate-resistant crops
e.g. tabacco, oil seed rape,roundup ready soybean
*Gentically modified to be resistant to herbicides coontaining glyphosphate (e.g. roundup)
*Glyphosphate inhibits enzymes involved in amino acid synthesis
*Gene from bacterium agrobacterium coding for enzymes with the same function that are not affected by glyphosphate
*Herbicide resistant gene acts as marker gene
→Herbicides will have no effect on plant, only the weeds
What are the advantages of herbicide-resistant crops-glyphosphate-resistant crops?
*Can control/Kill weeds
→Reduce competition
→Increase yeild
*Less manual weeding needed
What are the diasdvantages of herbicide-resistant crops-Glyphosphate-resistant crops?
*GM production cost is expensive
→Does it outweigh benefits?It has only 1% success…
*Monopoly/too much power held by multinational companies
*people may avoid/refuse to buy for GM food
*No long term studies done on effects on human health
*Possible allergic reactions in humans/adverse effects on the immune system
Enviromental:
1. Intensive use of herbicides
→High toxicity of more herbicide left after use
→Harmful to humans
- Cross pollination with wild plants/organic crops
→Result in new more resistant weeds=”superweeds”
→Superweeds are selected for by herbicide use
→Outcompete and lose traditional varieties of plants
→Loss of genetic diversity
→Cause effect on rest of food chain=less food or shelter for other species
Solution:Use sterile seeds/plants to avoid superweeds
Economic:
*High cost of GM seeds
→Seeds are sterile and canot use own seed
→Too expensie for people to buy/farmers to sell
→Diffucult for farmers in developing countries to obtain GM seed
→Might reduce efforts to relieve poverty
→Under-developed countries become more dependant on other countries
*Cost of herbicides:
→Cost of problems with pollution
→Cost of human health problems
→Loss due to contaminated crops of organic farms
What is the function of gel electrophoresis?
Function: To separate fragments of DNA according to length OR to seperate diff proteins according to mass/charge.
What are the components and their functions which are required in gel electrophoresis?
- Agarose gel (DNA) or Polyacrylamide gel (proteins) with wells:
Diff gel, diff impedance
Even spaces between the gel molecules so DNA/protein can move within gel properly
Wells to load DNA/Protein - Electrodes + electric feild:
Molecules with net negaive charge will move towards positive electrode - Buffer solution
mantain constant pH - Loading dye
Visible dye to track DNA migration
→Ensure electrophoresis is stopped before DNA/protein runs off the gel - Staining agent
DYE that stains the colourless DNA/protein
e.g. ethidium bromide causes DNA to fluoresce under UV light - DNA ladder or protein ladder
Has DNA fragments or susbtances of known lengths/mass
→Can estimate the length/mass of samples by comparing them to the ladder - Gene probes (DNA)
To locate a specific DNA sequence
Describe the procedure of Gel electrophoresis of DNA
- Obtain sufficiet quantity of DNA
*DNA can be amplified using PCR or gene cloning in bacteria - Cut DNA into fragments using restriction enzymes
*Many DNA fragments of diff lengths produced - DNA samples are mixed with loading dyes and staining agent
- Gel is covered with buffer solution
- DNA fragments are loaded into wells at the negative end of the gel (cathode)
*DNA ladder with DNA fragements of known length is usually run through the gel at the same time - A direct current is applied through the gel
- DNA is attracted towards the positive electrode (anode)
*Separation due to electric field/potential dfference
*Bcs DNA is negatively charged due to phosphate groups
*Gels acts a molecular seive
*Shorter fragments of DNA move faster and further per unit time through the gel than larger fragements
*Due to less impendance of gel
*DNA fragements seperate and arranged in order of size
8. Visualise bands under UV light
*Staining agent causes DN to fluoresce
*Each band is millions of DNA pieces of the same size
*Compare position with reference DNA to estimate length of fragements
9. DNA pieces transferred to membrane/nylon/nitrocellulose/absorbent paper
*This is called southern blotting
10. DNA heated to separate strands
11. DNA cooled and gene probes are added
*Gene probes are used to locate a specific DNA sequence or gene
*Gene probes=short, single stranded DNA (ssDNA)
*“Labelled”/attached with radioactive/fluorescent substance
*Can complementary base pair with specific DNA fragments
12. View DNA fragments under UV light/x-ray film
What is the procedure of genetic profilling/genetic fingerprinting?
- Extract DNA from tissue samples(e.g. blood, hair root, semen, saliva)
- Identity up to 13 VNTR regions
*Variable number of tandem repeats =short lengths of repetitive, non-coding sections of DNA
*Each induvidual has two allels for each VNTR
*Number of repeats differ between induviduals
*One from each homologous chromosome (from parents) - Quantity of DNA increased by polymerase chain reaction (PCR)
- DNA fragmented by restriction enzymes
- Gel electrophoresis of DNA→DNA pieces transferred to membrane →heated to seperate strands→gene probe added →Visualise under UV light/X-ray film→banding pattern seen
What are the applications of genetic fingerprinting/DNA profiling
Applications:
*Used to find relatedness with other individuals
*paternity testing
→Usually mother is known
→Child inherits one allele from mum, one from dad
*Criminal identification in forensics
What is genetic screening?
*Aka genetic testing
*Analysis of a person’s DNA to check for presence of a particular allele
*DNA obtained from tissue samples (e.g. blood)
*Can be carried out on embryo, fetid, newborn or adults
*Important examples: breast cancer, haemophilia, sickle cell anemia, Huntington’s disease, cystic fibrosis (CF), down syndrome
What are the genetic screening?
- Pre-implantation genetic diagnosis (PGD): Newly formed embryo from IVF, before implantation into uterus of mother.
- Prenatal screening: Unborn children/ fetus
3.Newborn screening: New born babies
- Carrier screening: Individuals with a family history of a genetic condition/cancer and potential parents
What is the procedure for pre-implantation genetic diagnosis (PGD)?
*Newly formed embryo from in vitro fertilisation (IVF)
*Tested before implamantation into uterus of mother
Procedure:
1)Embryo biopsy=removing a cell from an embryo
*At 8-cell stage (day 4/5)
2)PCR to amplify DNA
3)Gel electrophoresis
*Analyse DNA for faulty alleles to determine if embryo has genetic disease
*E.g. haemophilla huntingdon’s disease, cystic fibrosis
4)Pre-selection=select only embryo without faulty alleles for implantation
What are the social and ethical implications of pre-implantation genetic diagnosis (PGD)?
Social or ethical implications:
*Identify whether embryos from IVF have a genetic condition
→Avoid implantation of embryos with faulty alleles
→Allows couples to have children who would otherwise choose not to
But its controversial:
Ethical concerns:
*Embryos might be destroyed if not pre-selected for implantation
*Could leas to selection based on gender or specific traits (designer babies)
*Contrary to beliefs/values
*Genetic disease may not develop
What are the two methods to obtain tissue samples?
1) Amniocentesis:Needle used to withdraw foetal cells from amnitioc fluid
2) Chorionic villus sampling (CVS)-Sunction tube used to remove foetal cells from chorion (where plancenta will develop)
What are the advantages of prenatal screening?
Advantages:
*Helps to provide early diagnosis for fetuses in utero
→So can give early treatment when born
→Allow parents to prepare for the birth of a child who will need treatment for a considerable time or even throughout life
→Women can avoid late therapeutic abortions=terminate pregnancy for medical reasons
Describe newborn screening and carrier screening
E.g. haemophilla, sickle cell anaemia, cystic fibrosis, huntingdon’s disease down syndrome
E.g. breast cancer
*Faulty alleles of BRAC1 and BRAC2 genes
*Increase the chance of developing breast cancer
*High risk
If tested positive for the faulty alleles:
*Increased testing to detect cancer early
*Elective mastectomy=removal of breast(s) before the occurrence/diagnosis of cancer
What are the advantages of newborn screening and carrier screening?
Advantages:
*Can identify carriers of genetic conditions
→Allow couples who are both carriers of a genetic condition to choose not to have children
*Helps to provide early diagnosis
e.g. Huntington’s disease (symptoms only occur later in life)
→Can prepare for the future:
1. If present, enables lifestyle change/early treatment/regular check-ups
2. Prevantative treatment may be cheaper than treating disease itself
→If tested negative, genetic screening removes anxiety
What are the disadvantages of newborn screening and carrier screening?
Disadvantages:
*Test is expensive
*Many mutations still unknown so may still not lead to positive diagnosis
If person tested positive:
*For some diseases, no treatment is possible
*May lead to anxiety
*may experience social/financial discrimination (e.g. life insurance refusal)
*May still not develop diseases even though mutation is present
*Couple may decide to not hve children OR couple may be tested after they have children
Describe why a couple may be referred to genetic counselling and their job.
Couples are referred to genetic counsellors if they:
*Both have genetic disease in family or are carrier
*have history of recurrent miscarriages
*female is an older woman
Genetic conseller can:
*Run pedigree analysis + genetic screening
*Explain results of tests / estimate chances of having affected child
Genetic counsellor may discuss/advice on options:
*termination or therapeutic abortion
*therapies/treatments (e.g. gene therapy)
*The effect of having child on existing siblings
What is gene therapy?
*To treat serious, common genetic diseases
*Caused by a single faulty, recessive allele
*By inserting normal, dominant allele
*Into affected cells of induvidual
*Via a vector (e.g. virus, liposome or just naked DNA)
*Result=host will have recombinant DNA
Aim: To obtain functional normal polypeptide
*Reduce symptoms of disorder
*Restore/enhance cellular functions
*Increase quality of life/survival
e.g. SCID, cystic fibrosis, Leber’s congenital amaurosis (LCA), thalassemia, haemophillia
What happens if we try to do gene therapy on a faulty dominant allele?
If faulty allele was dominant, gene therapy is very very diffucult because…
*It will still be expressed even when the normal, recessive allele is present
*We’ll need to knowck out/remove/replace the faulty allel
→Extremely diffucult bcs it requires insertion of DNA into precise location in genome
e.g. huntington’s disease:
*Caused by a faulty, autosomal dominant allele of the huntintin gene
*Dominant allele also affects tissues in many parts of the body
→Gene therapy only alters genotype of a few targeted cells
Outline the procedure for gene therapy
procedure:
1. Obtain normal, dominant allele/cDNA from mRNA in cells of healthy person
2. Use gel electrophoresis+gene probe for identification
3. Use PCR to amplify DNA
4. Make recombinant DNA
*Use restriction enzyme to cut DNA and form complementary sticky ends
*Use DNA ligase to join cDNA with promoter/vector DNA
*Add human promoter upstream of targer allele to ensure transcription in host
5. Inset normal allele into vector
*Virus vector/liposome vector/naked DNA
6. Inject/spray into host
Describe the virus vector along with its problems and solution
- Virus vector
*Usually retroviruses
*Non-pathogenic
*Naturally inserts its viral DNA into host genome
*Recognises specific cells
Problems:
*May cause side effects/allergies
*May be removed by immune system before it reaches target cells
*The virus may trigger an immune response which destroys the infected cells
*Most non-pathogenic viruses are not very good at getting into cel, so very few cells receive the allele
*Short term effect as host cells have short lifespan
*Repeat treatments needed
*Retroviruses also cause random insertion of genes into host’s genome
*May inset its genes with another gene or within regulatory sequence
*May activate oncogenes/switch off tumore suppressor genes→cancer
*May have uncontrolled viral replication
Solution:
*Adeno-associated virus (AAV) does not insert genes into hosy’s genome, not passed on to daughter cells
*Lentiviruses can be modified to have no uncontrolled viral replication
*Or use different vector!
describe the liposome vector along with its problems
*Small sphere of phospholipid
*Sprayed as an aerosol/delivered using inhaler
*Liposome fuses with host cell surface membrane
*Trigger the immune response less than viruses
Problems:
*Not as effective in insertion as viruses
*Short term effect as host cells have short lifespan
*Repeat treatments needed
Describe the DNA vector along with its problems
*Very cheap
*Can be delivered via direct injection or gene gun
*No problems associated with using vectors
*Does not trigger immune response
Problems:
*Easily degraded
*Gene expression is very low
Describe severe combined immunodeficiency (SCID) and how gene therapy plays a role
*Severe combined immunodeficiency (SCID)
*Immune cells do not work properly
*Highly susceptible to infections
*B and T cells unable to make adenosine deaminase (ADA)
*Due to faulty allele coding for the enzyme
*X-linked recessive allele
Gene therapy:
1. Remove T lymphocytes
2. Introduce normal allele for ADA enzyme using virus
3. Replace T-lymphocytes into body
Problem: Regular transfusions of T cells are needed (not a permanent cure)
Alternative, long lasting method:
*Remove stem cells from bone marrow
*Insert normal alleles into stem cells using retroviruses
*Return stem cells into patient
Problem:
*Side effect=leukemia
*Due to random insertion of alleles into cell’s genome
Solution:
*Use Adeno-associated viruses (AAV)
What is leber’s congenital amaurosis (LCA) and what role does gene therapy play?
*Leber’s congenital amaurosis (LCA)
*Autosomal recessive eye disease
*Retina cells die off gradually
*Severe loss of vision at birth
*delivery of dominant, normal allele using adeno-associated virus (AAV)
*Vectors are injected directly into the retina
*So retina cells can make functional protein and restore vision
Suitable for treatment using gene therap bcs:
*Caused by recessive allele of a single gene
*Only need to get allele into a few cells
*Ease of access to affected area
*Only targer eye/no surgery needed
*Serious so worth the risk
What is cystic fibrosis?
*Inherited genetic disease
*faulty, autosomal recessive allelle of the CFTR gene
Normal CFTR protein:
*Transmembrane protein at cell surface membrane
*Acts as chloride channel
*Has binding site for ATP
*Cl- moves out of cell via active transport
*Water is drawn out from cell
→Normal/less viscous mucus formed
→Easy removal by cillia
Faulty CFTR allele:
a)Base deletion→Faulty CFTR (most common)
b) Base substition→STOP codon→Incomplete CFTR
faulty CFTR protein:
*No functional channels for Cl- ions
*Cl- ions do not move out
*Less water leaves cell
→Formation of thick, sticky mucus on cell surface membrane
→Cannot be removed by cilia
What are symptoms of cystic fibrosis?
*Thick and sticky mucus produced at lungs
*Mucus not moved effectively by cilia→mucus accumulates
*Mucus traps bacteria→more infections
*reduced gaseous exchange→due to longer diffusion pathway
*Difficulty in breathing, wheezing
*Coughing→cause lungs to be scarred
*Blocked pancreatic duct→reduced digestion, damage of pancreatic tissues causes diabetes
*Blocked sperm ducts/oviducts →reduced fertility
How can gene therapy help with cystsic fibrosis?
Traditional treatments:
*Deals with symptoms only not causes
1. Thick mucus in lungs→physiotherapy, percussion therapy to loosen and remove the mucus easily
2. recurrent bacterial infections→antibiotics
3. Reduced digestion→Enzyme supplements
Gene therapy:
*Insert normal allele for CFTR into lung cells
→usually inhaled or sprayed
*treats the cause rather than the symptoms
*no physiotherapy, antibiotics etc. needed
*Less time consuming than other treatments *Effects are short-lived (few days) and treatmet needs repeating
*May have side effects
What are the vectors commonly used for gene therapy in cystic fibrosis?
Vectors commonly used:
1)Liposomes
*Sprayed into nose
*not long lasting and only a few cells received the normal allele
2)Adenoviruses
*Normal harmless
*Used to infect respiratory cells
*Not all cells take up virus
*Side effects due to viral infection
What is the function of microarrays?
1) To distingush between allels of a gene
*Use DNA microarray
*Compare alleles found in genome of 2 induviduals/species
2)To identify expressed/transcribed genes
*Transcription of a gene produces mRNA
*So can assess gene expression by measuring mRNA levels
*Compares relative mRNA levels between 2 samples
*One acts as a control (normal cells)
What are the components required for microarrays?
Components: A microarray chip
*Involves a “chip” wich has >10,000 cells
*Gene probe is bound at known positions to a chip
*Gene probe=short lengths of single-stranded DNA (ssDNA) complementary to allele/gene
*Many copies of one type of probe placed in one cell of the microarray
*Diff cells has a diff type of gene probe complementary to specific allele/gene
*So many genes can be assessed at the same time!
What is the procedure by which microarrays work?
1A. To distinguish between alleles of a gene
Extract DNA from 2 samples
*Cut into DNA fragments
*Denature DNA into ssDNA by heating it
OR
1B. To identify expressed/transribed genes isolate mRNA of cells of 2 samples
*Use mRNA as a template to form single-stranded complementary DNA (cDNA)
*use reverse transcriptase
- Label ssDNA to hybridise/bind to the complementary gene probes
- Labelled ssDNA from both samples are washed over microarray
*Allow ssDNA to hybridise/bind to the complementary gene probes
*Unbound ssDNA will be washed off
*Bound DNA will not be washed off - Use laser/UV light to identify fluorescent spots
*Fluorescence=hybridised probes with ssDNA=gene is expressed
*Position and intensity is recorded by scanner
→Positions are then identified as named genes
*Intensity porportional to gene expression
What are the applications of microarrays?
Applications:
*genetic screening-can distinguish between alleles of a gene
*Drug testing-identify which genes the drugs have acted on
*Identifiying genes that are overexpressed/ not expressed in diseases
What is bioinformatics?
*Interdisciplinary field
*Collection, processing and analysis of biological information and data
*Using computer programs/software
*It is a store/database
→Collection of huge-quantities of data (“big data”) from all over the world
→From DNA sequencing, microarrays, other techniques
→Can search online for DNA and amino acid sequences
→And use that to model/predict tertiary structure of protein
Function: to allow comparison of DNA and protein sequences
What is DNA sequencing?
*Determine the exact sequence of bases in genes
*Fully automated process
*Genomes of many species have been published
*Gives information about the location of genes
*Provides evidence for the evolutionary links between organisms
What are the applications of bioinformatics?
Applications:
*Identify evolutionary relationships/genetic relatedness
*By Comparing between genomes/protein sequence of diff species
*Close similarities=recent common ancestry
*Compare beween genes/proteins of humans and model organism
*E.g. mouse, drosophila fruit flies, yeast, C. elegans roundworm
*If target gene have similar sequence and protein have similar structure
*Model organism can be used to investigate when and where genes have effect in humans
*Identify genome sequence and proteins of pathogens
*E.g. Plasmodium, straphylococcus areus
*Can help in vaccine/drug development
How can bioinformatics be used for drug development for malaria?
Can use database to:
*Identify genes
*Predict amino acid sequences of proteins
*Predict 3D structure of proteins
*Identify functions of proteins from 3D structure
Can formulate drug to:
*Bind with protein/enzyme
*Block activity of protein/enzyme
*Distrupt structure of protein/enzyme
*Prevent transcription of gene
what is the function of CRISPR-Cas9?
To cut DNA at specific loci and allow genome editing
Describe CRISPR-Cas9?
*Inexpensive, much easier and very effective
*Bacterial system to protect itself agaisnt virus
Components:
*Cas9=enzymes that cut DNA
*gRNA=guide RNA that is complementary to target DNA
Result:A double-stranded break is introduced at specific loci
*Cells will try to repair the double stranded break but repair is error prone
*Extra bases are included→gene is disrupted and deactivated
Potential:
*Technology is versatile and improving
→Can combine with other enzymes in order to introduce specific mutations/increase transcription/insert gene at specific location etc
→Can combine with GFP and act like a gene probe in situ
*Cure viral diseases and genetic diseases even cancer!
→Can inject CRISPR into body
→Extend life expectancy
Potenital issues:
*Increased preselectiom
*Designer babies, supersoldiers and biological weapons
