Gene Technology Flashcards

Question

Cut target gene and plasmid DNA (E.g. production of human insulin)

Answer 1

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

Answer 2

Recognize, bind and cut DNA at a specific sequence, called a restriction site * Hydrolyses/cleaves the phosphodiester bond btwn nucleotides * Diff kind of RE cuts at a diff specific seq * Most restriction sites are palindromic sequences * Restriction enzymes produces 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 pair At ‘blunt ends’ * NO unpaired nucleotides

Answer 3

For restriction enzyme to cut and produce sticky ends, so gene of interest can be inserted * Can have multiple to be cut by diff RE → has multiple cloning sites

Answer 4

Initiates transcription Allows binding of RNA polymerase/transcription factors Ensures correct strand 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 presence of lactose

Answer 5

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

Answer 6

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!

Answer 7

Mix cDNA/insulin gene with plasmid DNA ligase seals nicks in sugar-phosphate backbone Catalyze the formation of phosphodiester bond to form recombinant DNA

Answer 8

Recombinant plasmids mixed with bacteria treat bacteria with a solution of Ca+2 ions and allow to cool apply heat shock k (~42C) or use electroporation to increase chances of plasmids passing through cell surface membrane only 1% bacteria will take up recombinant plasmids and transform

Answer 9

Marker genes in the plasmid helps to recognise recombinant plasmids/modified bacteria/transgenic organisms * E.g. genes for antibiotic resistance, genes for fluorescent/easily stained substances * Gene of interest is inserted close to or into marker gene * Both target gene and marker gene are expressed

Answer 10

Bacteria did not take up recombinant plasmid Gene of interest did not join with plasmid, Cut plasmid just rejoined with itself

Answer 11

Use antibiotic selection (ampr, tetr) Use green fluorescent protein (GFP) Use an easily stained substance (β galactosidase, GUS)

Answer 12

Grow on agar containing the antibiotic bacteria with plasmid will be able to survive bacteria without plasmid dies Then make a replica plate by using sponge/velvet pad Grow bacteria on agar containing 2nd antibiotic (tetracycline) Bacteria with recombinant plasmid will die

Answer 13

Risk the spread of antibiotic resistance to other bacteria of same/diff species Plasmids are easily transferred between bacteria via conjunction this makes the use of antibiotics less effective in disease causing bacteria slower process for identification of transformed bacteria as well

Answer 14

Gene for fluorescent substances used as marker E.g. gene for Green fluorescent protein (GFP) * From jellyfish * GFP emits bright green light * When exposed to UV * Bacteria with plasmid will express GFP and will appear green

Answer 15

Use gene that codes for easily stained substances as marker E.g. gene for β galactosidase aka lacZ gene E.g. gene for GUS enzyme * Enzyme splits a special non-blue substrate into product that is blue * Bacteria with plasmid will become blue

Answer 16

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

Answer 17

Allow transformed bacteria cells to multiply/clone * Grow in large-scale culture / fermenter * E. coli can divide once every 20mins * 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

Answer 18

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 affects → less / no risk of allergic reaction 2. Effective in people who have developed tolerance to animal-derived insulin 3. Avoid ethical issues related to religion & 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 round 6. Less risk of contamination/infection → no risk of transfer of disease 7. Potential to engineer / improve recombinant proteins

Answer 19

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, insect larvae cells, mammalian cells

Answer 20

Human insulin Diabetes Recombinant bacteria Factor VIII -Helps blood clot in haemophilia patients -GM hamster kidney and ovary cells (in fermenter) Adenosine deaminase (ADA) -Help in development of B & T cells in severe combined immunodeficiency disorder (SCID) patients -GM insect larva of the cabbage looper moth

Answer 21

The organism with any altered DNA = Genetically modified organism (GMO) / transgenic organism * GMOs have recombinant DNA (rDNA) = combination of DNA from two or more sources * Genes to be transferred into an organism may be: 1. Extracted DNA from donor organism→transferred into another organism 2. Synthesised from mRNA of donor organism → converted to DNA → transferred 3. Synthesised chemically from nucleotides (in lab!) → transferred

Answer 22

Livestock can be engineered to: * Have high growth rate * Grow larger * Have higher yield (e.g. milk, meat) Crop plants can be engineered to: * Have higher yield * 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. high salinity, dry, hot, cold climates) * Grow in poor quality land, require less fertilizer → More cost effective / have health benefits → Less effect on food chain / pollinators

Answer 23

Much faster results * Allows the retention of other desirable characteristics of best varieties of species * Able to use best genes from other species of plants and even non-plants But… * Complicated process * Expensive * Not always successful

Answer 24

GM salmon * Insect resistance in Bt cotton * Herbicide resistance in soybean

Answer 25

non biodegradable chemicals entering the food chain as humans and animals consume the crops (Biomagnification/ Bio accumulation) useful insects get killed as well prevent affecting the non target species in the same environment

Answer 26

BT cotton protected against bool weevils gene of BT toxin from bacteria (bacillus thuringiensis) inserted into maize/cotton lethal insects but not other animals crop able to produce own insecticide only kills the target

Answer 27

Increased yields * Only kills specific insects that eats it and does not kill beneficial insects E.g. pollinators / bees / predators of pests → Conserve biodiversity / food web * Less pesticide used → Reduce risk of pesticide affecting other non-target species in the same environment * Less risk of harm to humans from spray drift / pesticide residues on food

Answer 28

Genetically modified to be resistant to herbicides containing glyphosate (e.g. Roundup) * Glyphosate inhibits enzymes involved in amino acid synthesis * Gene from bacterium Agrobacterium coding for enzymes with same function that are not affected by glyphosate * Herbicide resistant gene acts as marker gene → Herbicides will have no effect on plant, only the weeds

Answer 29

Can control/kill weeds → reduce competition → increase yield * Less manual weeding needed

Answer 30

intense use if herbicides high toxicity of more herbicide later after use harmful to humans cross pollination with wild plants results in more resistant weeds= super weeds [economic] Seeds are sterile and cannot use own seeds to expensive and difficult for farmers in developing countries under developed countries become more dependent on develop countries Hight cost of herbicides [cost of herbicide] cost of problems with pollution cost of human health problems loss due to contaminated crops of organic farms

Answer 31

* 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

Answer 32

Genes for growth hormone regulator transferred from diff species of salmon * Genes for promoter from another diff species Benefits: * High yield * Consistent yield all-year round * Conserve wild fish populations * All modified salmon eggs are triploid and sterile → so impossible for them to breed among themselves and with other salmon → eliminate impact to wild population

Answer 33

To separate fragments of DNA according to length Distinguish btwn alleles of a gene

Answer 34

Genetic fingerprinting / DNA profiling * Paternity testing * Criminal Investigations Genetic screening * Breast cancer (BRCA1, BRCA2) * Genes for haemophilia, SCA, Huntington’s Disease and CF Gene Therapy * Vectors (viruses, liposomes, naked DNA) * SCID, inherited eye disease, CF * Social and ethical considerations

Answer 35

Agarose gel with wells electrode+ electric field buffer solution Loading dye Staining agent DNA ladder Gene probes

Answer 36

1. Obtain sufficient quantity of DNA (DNA can be amplified using PCR or gene cloning in bacteria) 2. Cut DNA into fragments using restriction enzymes (Many DNA fragments of diff lengths produced) 3. DNA samples are mixed with loading dye and staining agent 4. Gel is covered with buffer solution 5. DNA fragments are loaded into wells at the negative end of the gel (cathode) (DNA ladder with DNA fragments of known lengths is usually run through the gel at the same time) 6. A direct current is applied through the gel 7 . DNA is attracted towards the positive electrode (anode) (Separation due to electric field/potential difference) (Bcs DNA is negatively charged due to the phosphate groups) DNA fragments separate and arranged in order of size 8. Visualize bands under UV light 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

Answer 37

pre implantation genetic diagnosis (PGD) when newly formed embryo from IVF before implantation into the uterus of the mother prenatal screening unborn child/ fetus newborn screening carrier screening (adulthood) family history with conditions or health issues

Answer 38

a 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, fetus, newborn or adults * Important examples: breast cancer, haemophilia, sickle cell anaemia, Huntington’s disease, cystic fibrosis (CF), Down syndrome

Answer 39

1. Extract DNA from tissue samples (e.g. blood, hair root, semen, saliva) 2. Identify up to 13 VNTR regions * Variable Number of Tandem Repeats (VNTR) = short lengths of repetitive, non-coding sections of DNA * Each individual has two alleles for each VNTR * Number of repeats differ between individuals * One from each homologous chromosome 3. Quantity of DNA increased by Polymerase Chain Reaction (PCR) 4. DNA fragmented by restriction enzymes 5. Gel electrophoresis of DNA → DNA pieces transferred to membrane → heated to separate strands → gene probe added → Visualize under UV light / X-ray film → banding pattern seen

Answer 40

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

Answer 41

Hormone treatment used to induce superovulation 2. Many oocytes harvested from female 3. Obtain fresh / frozen sperm 4. Oocytes mixed with sperm – Ideally genetically different 5. Conduct genetic test and select embryos that are most likely to survive 6. Embryo transfer! – Embryo implanted in uterus of donor female – OR may use similar species / non-rare breed as surrogate mother – OR freeze embryos and store for long time in frozen zoo

Answer 42

Newly formed embryo from in vitro fertilisation (IVF) * Tested before implantation into uterus of mother

Answer 43

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. haemophilia, Huntington’s disease, cystic fibrosis 4) Pre-selection = select only embryo without faulty alleles for implantation

Answer 44

[social] 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 [ethical] Embryos might be destroyed if not pre-selected for implantation * Could lead to selection based on gender or specific traits (“designer babies”) * Contrary to beliefs / values * Genetic disease may not develop

Answer 45

1. amniocentesis 2. chorionic VIIII sampling

Answer 46

Helps to provide early diagnosis for fetuses in utero → So can give early treatment when born → Allows 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

Answer 47

E.g. haemophilia, sickle cell anaemia, cystic fibrosis (CF), Huntington’s disease, Down syndrome E.g. breast cancer * Faulty alleles of the BRCA1 and BRCA2 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

Answer 48

Advantages * Can identify carriers of genetic conditions → Allows 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. preventative treatment may be cheaper than treating disease itself → if tested negative, genetic screening removes anxiety

Answer 49

test is expensive many mutations still unknown so may still not lead to positive diagnosis At some instances no treatment is found many experience social and financial discrimination may not develop diseases even though mutation present. couple decide to not continue the bloodline to after knowing the positive test

Answer 50

both have genetic disease (in family) or are carriers have history of recurrent miscarriages female is an older woman

Answer 51

run pedigree analysis + genetic screening * explain results of tests / estimates chances of having affected child * an provide couple with information on the risk of genetic diseases Genetic counsellor may discuss/advice on options: * termination or therapeutic abortion * therapies / treatments (e.g. gene therapy) * financial implications of having affected child * the effect of having affected child on existing siblings * other ethical issues

Answer 52

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 target allele to ensure transcription in host 5. Insert normal allele into vector * Virus vector / Liposome vector / naked DNA 6. Inject / spray into host

Answer 53

retroviruses also cause random insertion of genes into hosts genome may insert its gene within another gene or within regulatoru sequences may activate oncogens

Answer 54

1. Virus vector * Usually retroviruses * Non-pathogenic * Naturally inserts its viral DNA into host genome * Recognises specific cells

Answer 55

liposomes vectors small sphere of phospholipid sprayed as an aerosol/ delivered using an inhaler liposome fuses with host cell surface membrane The virus may trigger an immune response which destroys the infected cells Short term effect as host cells have short lifespan Repeat treatments needed

Answer 56

very cheap and can be delivered via direct injection or gene gun no problem associated with using vectors does not trigger immune system problems become it being easily degraded gene expression is very low

Answer 57

severe combined immunodeficiency immune cells do not function 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

Answer 58

1. Remove T lymphocytes 2. Introduce normal allele for ADA enzyme through a virus 3. Replace T-lymphocytes into body Problem: Regular transfusions of T cells are needed (not a permanent cure)

Answer 59

* Remove stem cells from bone marrow * Insert normal alleles into stem cells using retroviruses * Return stem cells into patient

Answer 60

Problem: * Side effect = leukemia * Due to random insertion of alleles into cell’s genome Solution: * Use Adeno-associated viruses (AAV)

Answer 61

leber's congenital (Born with a deformation) is an 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

Answer 62

Suitable for treatment using gene therapy 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 targets eye / no surgery needed * Serious so worth the risk

Answer 63

inherited genetic disease faulty, autosomal recessive allele of the CFTR gene Normal CFTR protein: transmembrane normal/ less viscous mucus formed easy removal by cilia faulty allele: no functional channels for cl- ions Cl- ions do not move out less water leaves cell formation of thick. sticky mucus on cell surface memebrane cannot be moved by cilia

Answer 64

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

Answer 65

Thick mucus in lungs → physiotherapy, percussion therapy to loosen & remove mucus easily Recurrent bacterial infections → antibiotics Reduced digestion → enzyme supplements

Answer 66

Insert normal allele for CFTR into lungs 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 treatment needs repeating * May have side-effects

Answer 67

Liposomes sprayed into the nose not long lasting and only a few cells received the normal allele adenoviruses harmless used to infect respiratory cells not all cells take up virus side effect due to viral infection

Answer 68

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)

Answer 69

Involves a “chip” with 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!

Answer 70

1. Isolate all mRNA of cells of 2 samples Use mRNA as a template to form single- stranded complementary DNA (cDNA) Use reverse transcriptase 2. Label ssDNA using fluorescent ‘tags’ * One sample red, one sample green 3. 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 4. Use laser/UV light to identify fluorescent spots fluorescence= hyberdised probes with ssDNA= gene is expressed position and intensity is recorded by scanner intensity proportional to gene expression

Answer 71

identify genes that are overexpressed or not expressed in diseases genetic screening- can distinguish between alleles of a gene drug testing- identify which genes that drugs have acted on

Answer 72

provides a large database used to find SNPs it is a fast and accurate software for statistical analysis

Answer 73

DNA analysis not accessible to everyone may result in symptoms like anxiety and panic predictions may not be accurate can effect an individuals job/ insurance an early treatment can leas to better planning for people

Answer 74

Ensembl – genomes of vertebrates ii) UniProt – primary sequences & functions of proteins iii) BLAST search tool – an algorithm to search the database for target DNA or protein sequence

Answer 75

* Identify evolutionary relationships / genetic relatedness * By comparing between genomes/protein seq of diff species * Close similarities = recent common ancestry * Compare between genes / proteins of humans and model organisms * 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 seq and proteins of pathogens * Can help in vaccine/drug development

Answer 76

Form of genetic engineering * Involving the insertion, deletion or replacement of DNA * At specific sites in the genome

Answer 77

Clustered Regularly Interspaced Short Palindromic Repeats → Part of the bacterial / archaea genome

Answer 78

Cas9 (CRISPR-associated enzyme) endonuclease enzyme has 2 active sites to cut DNA across both strands

Answer 79

A double-stranded break at specific loci * DNA repair mechanisms can repair the break by: 1. Adding one or more nucleotides → Extra bases are included → Gene is disrupted and deactivated 2. Inserting a short length of prepared double stranded DNA with a specific base sequence → Desired gene is modified and expressed * Inexpensive, easy and very effective way for gene editing

Answer 80

Bacteriophages (virus) invades bacteria → Bacteria takes some DNA from phages → Integrate into CRISPR DNA base sequences (“memory”) → CRISPR RNA is formed via transcription → Used as guide RNA (gRNA) = ~20bases complementary to target DNA → gRNA guides Cas9 to specific DNA sequence of the phages to cut DNA → Pass down to next gen

Answer 81

Cure diseases (viral, genetic, maybe cancer?) – Remove / switch off faulty alleles → no faulty protein made – Replace part of the faulty allele → make functioning protein * Very cool experiments and research → 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 → E.g. The bold plan to end malaria with a gene drive

Answer 82

Increased preselection → Designer babies * Super soldiers and biological weapons